RNA INTERFERENCE MEDIATED INHIBITION OF XIAP GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA)

ABSTRACT

This invention relates to compounds, compositions, and methods useful for modulating XIAP gene expression using short interfering nucleic acid (siNA) molecules. This invention also relates to compounds, compositions, and methods useful for modulating the expression and activity of other genes involved in pathways of XIAP gene expression and/or activity by RNA interference (RNAi) using small nucleic acid molecules. In particular, the instant invention features small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methods used to modulate the expression of XIAP genes.

This application is a continuation of U.S. patent application Ser. No.10/567,888, filed on Dec. 15, 2006, which is a 371 of PCT/US2004/025589,filed on Aug. 6, 2004, which claims benefit of U.S. ProvisionalApplication No. 60/493,561 filed Aug. 8, 2003; and parent U.S. patentapplication Ser. No. 10/567,888 is also a continuation-in-part ofInternational Patent Application No. PCT/US04/16390, filed May 24, 2004,which is a continuation-in-part of U.S. patent application Ser. No.10/826,966, filed Apr. 16, 2004, which is continuation-in-part of U.S.patent application Ser. No. 10/757,803, filed Jan. 14, 2004, which is acontinuation-in-part of U.S. patent application Ser. No. 10/720,448,filed Nov. 24, 2003, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/693,059, filed Oct. 23, 2003, which is acontinuation-in-part of U.S. patent application Ser. No. 10/444,853,filed May 23, 2003, which is a continuation-in-part of InternationalPatent Application No. PCT/US03/05346, filed Feb. 20, 2003, and acontinuation-in-part of International Patent Application No.PCT/US03/05028, filed Feb. 20, 2003, both of which claim the benefit ofU.S. Provisional Application No. 60/358,580 filed Feb. 20, 2002, U.S.Provisional Application No. 60/363,124 filed Mar. 11, 2002, U.S.Provisional Application No. 60/386,782 filed Jun. 6, 2002, U.S.Provisional Application No. 60/406,784 filed Aug. 29, 2002, U.S.Provisional Application No. 60/408,378 filed Sep. 5, 2002, U.S.Provisional Application No. 60/409,293 filed Sep. 9, 2002, and U.S.Provisional Application No. 60/440,129 filed Jan. 15, 2003. The instantapplication claims the benefit of all the listed applications, which arehereby incorporated by reference herein in their entireties, includingthe drawings.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 CFR§1.52(e)(5), is incorporated herein by reference. The sequence listingtext file submitted via EFS contains the file “SequenceListing60USCNT”,created on Sep. 4, 2008, which is 202,288 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of X-linked inhibitor ofapoptosis protein (XIAP) gene expression and/or activity. The presentinvention is also directed to compounds, compositions, and methodsrelating to traits, diseases and conditions that respond to themodulation of expression and/or activity of genes involved in XIAP geneexpression pathways or other cellular processes that mediate themaintenance or development of such traits, diseases and conditions.Specifically, the invention relates to small nucleic acid molecules,such as short interfering nucleic acid (siNA), short interfering RNA(siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), and shorthairpin RNA (shRNA) molecules capable of mediating RNA interference(RNAi) against XIAP gene expression. Such small nucleic acid moleculesare useful, for example, in providing compositions for treatment oftraits, diseases and conditions that can respond to modulation of XIAPexpression in a subject, such as those associated with the maintenanceand/or development of cancer and other proliferative disorders.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, Genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J., 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of an siRNAduplex is required for siRNA activity and that ATP is utilized tomaintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation of doublestranded RNA-dependent protein kinase PKR, specifically 2′-amino or2′-O-methyl nucleotides, and nucleotides containing a 2′-O or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certain singlestranded siRNA constructs, including certain 5′-phosphorylated singlestranded siRNAs that mediate RNA interference in HeLa cells. Harborth etal., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105,describe certain chemically and structurally modified siRNA molecules.Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically andstructurally modified siRNA molecules. Woolf et al., International PCTPublication Nos. WO 03/064626 and WO 03/064625 describe certainchemically modified dsRNA constructs.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating the expression of genes associated with inhibitor ofapoptosis proteins (IAPs), for example, XIAP (X-linked inhibitor ofapoptosis protein) and related genes, such as HIAP1 (human inhibitor orapoptosis 1), HIAP2 (human inhibitor or apoptosis 2), NAIP (neuronalapoptosis inhibitor protein) and other IAP's (inhibitors of apoptosisproteins) using short interfering nucleic acid (siNA) molecules. Thisinvention also relates to compounds, compositions, and methods usefulfor modulating the expression and activity of other genes involved inpathways of XIAP gene expression and/or activity by RNA interference(RNAi) using small nucleic acid molecules. In particular, the instantinvention features small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA), doublestranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA)molecules and methods used to modulate the expression of XIAP genes.

An siNA of the invention can be unmodified or chemically modified. AnsiNA of the instant invention can be chemically synthesized, expressedfrom a vector or enzymatically synthesized. The instant invention alsofeatures various chemically modified synthetic short interfering nucleicacid (siNA) molecules capable of modulating XIAP gene expression oractivity in cells by RNA interference (RNAi). The use of chemicallymodified siNA improves various properties of native siNA moleculesthrough increased resistance to nuclease degradation in vivo and/orthrough improved cellular uptake. Further, contrary to earlier publishedstudies, siNA having multiple chemical modifications retains its RNAiactivity. The siNA molecules of the instant invention provide usefulreagents and methods for a variety of therapeutic, diagnostic, targetvalidation, genomic discovery, genetic engineering, and pharmacogenomicapplications.

In one embodiment, the invention features one or more siNA molecules andmethods that independently or in combination modulate the expression ofXIAP, HIAP1, HIAP2, and/or NAIP gene(s) encoding proteins, such asproteins comprising XIAP, HIAP1, HIAP2, and/or NAIP associated with themaintenance and/or development of cancer and other proliferativedisorders, such as ovarian cancer; cancers of non-lymphoid parenchymalorgans including the heart, placenta, skeletal muscle and lung; breastcancer; cancers of the head and neck, including various lymphomas suchas mantle cell lymphoma; non-Hodgkins lymphoma; adenoma; squamous cellcarcinoma; laryngeal carcinoma; cancers of the retina; cancers of theesophagus; multiple myeloma; melanoma; colorectal cancer; lung cancer;bladder cancer; prostate cancer; glioblastoma; and proliferativediseases and conditions such as restenosis and polycystic kidneydisease; and any other indications that can respond to the level of aXIAP gene in a cell or tissue, for example, genes encoding sequencescomprising those sequences referred to by GenBank Accession Nos. shownin Table I, referred to herein generally as XIAP. The description belowof the various aspects and embodiments of the invention is provided withreference to exemplary XIAP gene referred to herein as XIAP, which isalso known as BIRC4. However, the various aspects and embodiments arealso directed to other apoptosis inhibitor genes such as HIAP1, HIAP2,and NAIP, and other XIAP genes, such XIAP homolog genes, XIAP transcriptvariants and polymorphisms (e.g., single nucleotide polymorphism,(SNPs)) associated with certain XIAP genes, including genes encoding anyXIAP ligands and receptors. As such, the various aspects and embodimentsare also directed to other genes, such as HIAP1, HIAP2, AND NAIP, thatare involved in XIAP mediated pathways of signal transduction or geneexpression that are involved in, for example, the progression,development, and/or maintenance of disease, such as cancer and otherproliferative disorders (e.g., ovarian cancer; cancers of non-lymphoidparenchymal organs including the heart, placenta, skeletal muscle andlung; breast cancer; cancers of the head and neck, including variouslymphomas such as mantle cell lymphoma; non-Hodgkins lymphoma; adenoma;squamous cell carcinoma; laryngeal carcinoma; cancers of the retina;cancers of the esophagus; multiple myeloma; melanoma; colorectal cancer;lung cancer; bladder cancer; prostate cancer; glioblastoma; andproliferative diseases and conditions such as restenosis and polycystickidney disease; and any other indications that can respond to the levelof a XIAP gene in a cell or tissue). These additional genes can beanalyzed for target sites using the methods described for XIAP genesherein. Thus, the modulation of other genes and the effects of suchmodulation of the other genes can be performed, determined, and measuredas described herein.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene, wherein said siNA molecule comprises about 15 to about28 base pairs.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a XIAPRNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 28 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the XIAP RNA for the siNA molecule to direct cleavageof the XIAP RNA via RNA interference, and the second strand of said siNAmolecule comprises nucleotide sequence that is complementary to thefirst strand.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of a XIAPRNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first and a second strand, each strand of the siNAmolecule is about 18 to about 23 nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the XIAP RNA for the siNA molecule to direct cleavageof the XIAP RNA via RNA interference, and the second strand of said siNAmolecule comprises nucleotide sequence that is complementary to thefirst strand.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a XIAP RNA via RNA interference (RNAi), wherein eachstrand of the siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the XIAP RNA for the siNAmolecule to direct cleavage of the XIAP RNA via RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a XIAP RNA via RNA interference (RNAi), wherein eachstrand of the siNA molecule is about 18 to about 23 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the XIAP RNA for the siNAmolecule to direct cleavage of the XIAP RNA via RNA interference.

In one embodiment, the invention features an siNA molecule thatdown-regulates expression of a XIAP gene, for example, wherein the XIAPgene comprises XIAP encoding sequence. In one embodiment, the inventionfeatures an siNA molecule that down-regulates expression of a XIAP gene,for example, wherein the XIAP gene comprises XIAP non-coding sequence orregulatory elements involved in XIAP gene expression.

In one embodiment, an siNA of the invention is used to inhibit theexpression of XIAP genes or a XIAP gene family, wherein the genes orgene family sequences share sequence homology. Such homologous sequencescan be identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs, that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering XIAP targets that share sequence homology. As such, oneadvantage of using siNAs of the invention is that a single siNA can bedesigned to include nucleic acid sequence that is complementary to thenucleotide sequence that is conserved between the homologous genes. Inthis approach, a single siNA can be used to inhibit expression of morethan one gene instead of using more than one siNA molecule to target thedifferent genes.

In one embodiment, the invention features an siNA molecule having RNAiactivity against XIAP RNA, wherein the siNA molecule comprises asequence complementary to any RNA having XIAP encoding sequence, such asthose sequences having GenBank Accession Nos. shown in Table I. Inanother embodiment, the invention features an siNA molecule having RNAiactivity against XIAP RNA, wherein the siNA molecule comprises asequence complementary to an RNA having variant XIAP encoding sequence,for example other mutant XIAP genes not shown in Table I but known inthe art to be associated with the maintenance and/or development ofdevelopment of cancer and other proliferative disorders, such as ovariancancer; cancers of non-lymphoid parenchymal organs including the heart,placenta, skeletal muscle and lung; breast cancer; cancers of the headand neck, including various lymphomas such as mantle cell lymphoma;non-Hodgkins lymphoma; adenoma; squamous cell carcinoma; laryngealcarcinoma; cancers of the retina; cancers of the esophagus; multiplemyeloma; melanoma; colorectal cancer; lung cancer; bladder cancer;prostate cancer; glioblastoma; and proliferative diseases and conditionssuch as restenosis and polycystic kidney disease; and any otherindications that can respond to the level of a XIAP gene in a cell ortissue. Chemical modifications as shown in Tables III and IV orotherwise described herein can be applied to any siNA construct of theinvention. In another embodiment, an siNA molecule of the inventionincludes a nucleotide sequence that can interact with nucleotidesequence of a XIAP gene and thereby mediate silencing of XIAP geneexpression, for example, wherein the siNA mediates regulation of XIAPgene expression by cellular processes that modulate the chromatinstructure or methylation patterns of the XIAP gene and preventtranscription of the XIAP gene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of XIAP proteins arising from XIAPhaplotype polymorphisms that are associated with a disease or condition,(e.g., cancer, such as ovarian cancer; cancers of non-lymphoidparenchymal organs including the heart, placenta, skeletal muscle andlung; breast cancer; cancers of the head and neck, including variouslymphomas such as mantle cell lymphoma; non-Hodgkins lymphoma; adenoma;squamous cell carcinoma; laryngeal carcinoma; cancers of the retina;cancers of the esophagus; multiple myeloma; melanoma; colorectal cancer;lung cancer; bladder cancer; prostate cancer; glioblastoma; andproliferative diseases and conditions such as restenosis and polycystickidney disease). Analysis of XIAP genes, or XIAP protein or RNA levelscan be used to identify subjects with such polymorphisms or thosesubjects who are at risk of developing traits, conditions, or diseasesdescribed herein. These subjects are amenable to treatment, for example,treatment with siNA molecules of the invention and any other compositionuseful in treating diseases related to XIAP gene expression. As such,analysis of XIAP protein or RNA levels can be used to determinetreatment type and the course of therapy in treating a subject.Monitoring of XIAP protein or RNA levels can be used to predicttreatment outcome and to determine the efficacy of compounds andcompositions that modulate the level and/or activity of certain XIAPproteins associated with a trait, condition, or disease.

In one embodiment of the invention an siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a XIAP protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a XIAP gene or a portion thereof.

In another embodiment, an siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a XIAP protein or a portion thereof. The siNA moleculefurther comprises a sense region, wherein said sense region comprises anucleotide sequence of a XIAP gene or a portion thereof.

In another embodiment, the invention features an siNA moleculecomprising a nucleotide sequence in the antisense region of the siNAmolecule that is complementary to a nucleotide sequence or portion ofsequence of a XIAP gene. In another embodiment, the invention featuresan siNA molecule comprising a region, for example, the antisense regionof the siNA construct, complementary to a sequence comprising a XIAPgene sequence or a portion thereof.

In one embodiment, the antisense region of XIAP siNA constructscomprises a sequence complementary to sequence having any of SEQ ID NOs.1-467 or 935-942. In one embodiment, the antisense region of XIAPconstructs comprises sequence having any of SEQ ID NOs. 468-934,951-958, 967-974, 983-990, 999-1006, 1015-1038, 1040, 1042, 1044, 1047,1049, 1051, 1053, or 1056. In another embodiment, the sense region ofXIAP constructs comprises sequence having any of SEQ ID NOs. 1-467,935-950, 959-966, 975-982, 991-998, 1007-1014, 1039, 1041, 1043, 1045,1046, 1048, 1050, 1052, 1054, or 1055.

In one embodiment, an siNA molecule of the invention comprises any ofSEQ ID NOs. 1-1056. The sequences shown in SEQ ID NOs. 1-1056 are notlimiting. An siNA molecule of the invention can comprise any contiguousXIAP sequence (e.g., about 15 to about 25 or more, or about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25 or more contiguous XIAP nucleotides).

In yet another embodiment, the invention features an siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in Table I.Chemical modifications in Tables III and IV and described herein can beapplied to any siNA construct of the invention.

In one embodiment of the invention an siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a RNA sequence or aportion thereof encoding a XIAP protein, and wherein said siNA furthercomprises a sense strand having about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, and wherein said sense strand and said antisense strand aredistinct nucleotide sequences where at least about 15 nucleotides ineach strand are complementary to the other strand.

In another embodiment of the invention an siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a RNAsequence encoding a XIAP protein, and wherein said siNA furthercomprises a sense region having about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein said sense region and said antisense region arecomprised in a linear molecule where the sense region comprises at leastabout 15 nucleotides that are complementary to the antisense region.

In one embodiment, an siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a XIAP gene. Because XIAPgenes can share some degree of sequence homology with each other, siNAmolecules can be designed to target a class of XIAP genes or alternatelyspecific XIAP genes (e.g., polymorphic variants) by selecting sequencesthat are either shared amongst different XIAP targets or alternativelythat are unique for a specific XIAP target. Therefore, in oneembodiment, the siNA molecule can be designed to target conservedregions of XIAP RNA sequences having homology among several XIAP genevariants so as to target a class of XIAP genes with one siNA molecule.Accordingly, in one embodiment, the siNA molecule of the inventionmodulates the expression of one or both XIAP alleles in a subject. Inanother embodiment, the siNA molecule can be designed to target asequence that is unique to a specific XIAP RNA sequence (e.g., a singleXIAP allele or XIAP single nucleotide polymorphism (SNP)) due to thehigh degree of specificity that the siNA molecule requires to mediateRNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response are doublestranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 15 to about 30 base pairs between oligonucleotidescomprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2,or 3) nucleotides, for example, about 21-nucleotide duplexes with about19 base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs. In yet another embodiment, siNA molecules ofthe invention comprise duplex nucleic acid molecules with blunt ends,where both ends are blunt, or alternatively, where one of the ends isblunt.

In one embodiment, the invention features one or more chemicallymodified siNA constructs having specificity for XIAP expressing nucleicacid molecules, such as RNA encoding a XIAP protein. In one embodiment,the invention features a RNA based siNA molecule (e.g., an siNAcomprising 2′-OH nucleotides) having specificity for XIAP expressingnucleic acid molecules that includes one or more chemical modificationsdescribed herein. Non-limiting examples of such chemical modificationsinclude without limitation phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, “universal base” nucleotides, “acyclic” nucleotides,5-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxyabasic residue incorporation. These chemical modifications, when used invarious siNA constructs, (e.g., RNA based siNA constructs), are shown topreserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds. Furthermore, contraryto the data published by Parrish et al., supra, applicant demonstratesthat multiple (greater than one) phosphorothioate substitutions arewell-tolerated and confer substantial increases in serum stability formodified siNA constructs.

In one embodiment, an siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, and/or bioavailability. For example, ansiNA molecule of the invention can comprise modified nucleotides as apercentage of the total number of nucleotides present in the siNAmolecule. As such, an siNA molecule of the invention can generallycomprise about 5% to about 100% modified nucleotides (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides). The actual percentageof modified nucleotides present in a given siNA molecule will depend onthe total number of nucleotides present in the siNA. If the siNAmolecule is single stranded, the percent modification can be based uponthe total number of nucleotides present in the single stranded siNAmolecules. Likewise, if the siNA molecule is double stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands.

One aspect of the invention features a double stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a XIAPgene. In one embodiment, the double stranded siNA molecule comprises oneor more chemical modifications and each strand of the double strandedsiNA is about 21 nucleotides long. In one embodiment, the doublestranded siNA molecule does not contain any ribonucleotides. In anotherembodiment, the double stranded siNA molecule comprises one or moreribonucleotides. In one embodiment, each strand of the double strandedsiNA molecule independently comprises about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein each strand comprises about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to the nucleotides of the otherstrand. In one embodiment, one of the strands of the double strandedsiNA molecule comprises a nucleotide sequence that is complementary to anucleotide sequence or a portion thereof of the XIAP gene, and thesecond strand of the double stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence ofthe XIAP gene or a portion thereof.

In another embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene comprising an antisense region, wherein the antisenseregion comprises a nucleotide sequence that is complementary to anucleotide sequence of the XIAP gene or a portion thereof, and a senseregion, wherein the sense region comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the XIAP gene or aportion thereof. In one embodiment, the antisense region and the senseregion independently comprise about 15 to about 30 (e.g. about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense region comprises about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.

In another embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the XIAP geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region.

In one embodiment, an siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, an siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 28” (Table IV) or any combination thereof(see Table IV)) and/or any length described herein can comprise bluntends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, an siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule. The sense region can be connected to the antisense regionvia a linker molecule, such as a polynucleotide linker or anon-nucleotide linker.

In one embodiment, the invention features double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene, wherein the siNA molecule comprises about 15 to about 30(e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30) base pairs, and wherein each strand of the siNA moleculecomprises one or more chemical modifications. In another embodiment, oneof the strands of the double stranded siNA molecule comprises anucleotide sequence that is complementary to a nucleotide sequence of aXIAP gene or a portion thereof, and the second strand of the doublestranded siNA molecule comprises a nucleotide sequence substantiallysimilar to the nucleotide sequence or a portion thereof of the XIAPgene. In another embodiment, one of the strands of the double strandedsiNA molecule comprises a nucleotide sequence that is complementary to anucleotide sequence of a XIAP gene or portion thereof, and the secondstrand of the double stranded siNA molecule comprises a nucleotidesequence substantially similar to the nucleotide sequence or portionthereof of the XIAP gene. In another embodiment, each strand of the siNAmolecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and eachstrand comprises at least about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides thatare complementary to the nucleotides of the other strand. The XIAP genecan comprise, for example, sequences referred to in Table I.

In one embodiment, an siNA molecule of the invention comprises noribonucleotides. In another embodiment, an siNA molecule of theinvention comprises ribonucleotides.

In one embodiment, an siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a XIAP gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the XIAP gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g. about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.The XIAP gene can comprise, for example, sequences referred to in TableI. In another embodiment, the siNA is a double stranded nucleic acidmolecule, where each of the two strands of the siNA moleculeindependently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36,37, 38, 39, or 40) nucleotides, and where one of the strands of the siNAmolecule comprises at least about 15 (e.g. about 15, 16, 17, 18, 19, 20,21, 22, 23, 24 or 25 or more) nucleotides that are complementary to thenucleic acid sequence of the XIAP gene or a portion thereof.

In one embodiment, an siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a XIAP gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. The XIAP genecan comprise, for example, sequences referred in to Table I.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the XIAP geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thesiNA molecule has one or more modified pyrimidine and/or purinenucleotides. In one embodiment, the pyrimidine nucleotides in the senseregion are 2′-O-methylpyrimidine nucleotides or 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-O-methyl purine nucleotides. In another embodiment, thepyrimidine nucleotides in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides. In one embodiment, thepyrimidine nucleotides in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides and the purine nucleotides present in theantisense region are 2′-O-methyl or 2′-deoxy purine nucleotides. Inanother embodiment of any of the above-described siNA molecules, anynucleotides present in a non-complementary region of the sense strand(e.g. overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule, and wherein the fragment comprising the sense regionincludes a terminal cap moiety at the 5′-end, the 3′-end, or both of the5′ and 3′ ends of the fragment. In one embodiment, the terminal capmoiety is an inverted deoxy abasic moiety or glyceryl moiety. In oneembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In anotherembodiment, each of the two fragments of the siNA molecule independentlycomprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39,or 40) nucleotides. In a non-limiting example, each of the two fragmentsof the siNA molecule comprise about 21 nucleotides.

In one embodiment, the invention features an siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide. The siNA can be, for example, about 15 toabout 40 nucleotides in length. In one embodiment, all pyrimidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro pyrimidinenucleotides. In one embodiment, the modified nucleotides in the siNAinclude at least one 2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluorouridine nucleotide. In another embodiment, the modified nucleotides inthe siNA include at least one 2′-deoxy-2′-fluoro cytidine and at leastone 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, alluridine nucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a method of increasing thestability of an siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-deoxy-2′-fluoro cytidine and at least one2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all uridinenucleotides present in the siNA are 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all cytidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro cytidine nucleotides. In one embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In one embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In one embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene comprising a sense region and an antisense region,wherein the antisense region comprises a nucleotide sequence that iscomplementary to a nucleotide sequence of RNA encoded by the XIAP geneor a portion thereof and the sense region comprises a nucleotidesequence that is complementary to the antisense region, and wherein thepurine nucleotides present in the antisense region comprise2′-deoxy-purine nucleotides. In an alternative embodiment, the purinenucleotides present in the antisense region comprise 2′-O-methyl purinenucleotides. In either of the above embodiments, the antisense regioncan comprise a phosphorothioate internucleotide linkage at the 3′ end ofthe antisense region. Alternatively, in either of the above embodiments,the antisense region can comprise a glyceryl modification at the 3′ endof the antisense region. In another embodiment of any of theabove-described siNA molecules, any nucleotides present in anon-complementary region of the antisense strand (e.g. overhang region)are 2′-deoxy nucleotides.

In one embodiment, the antisense region of an siNA molecule of theinvention comprises sequence complementary to a portion of a XIAPtranscript having sequence unique to a particular XIAP disease relatedallele, such as sequence comprising a single nucleotide polymorphism(SNP) associated with the disease specific allele. As such, theantisense region of an siNA molecule of the invention can comprisesequence complementary to sequences that are unique to a particularallele to provide specificity in mediating selective RNAi against thedisease, condition, or trait related allele.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a XIAP gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments wherein one fragment comprises the senseregion and the second fragment comprises the antisense region of thesiNA molecule. In another embodiment, the siNA molecule is a doublestranded nucleic acid molecule, where each strand is about 21nucleotides long and where about 19 nucleotides of each fragment of thesiNA molecule are base-paired to the complementary nucleotides of theother fragment of the siNA molecule, wherein at least two 3′ terminalnucleotides of each fragment of the siNA molecule are not base-paired tothe nucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acidmolecule, where each strand is about 19 nucleotide long and where thenucleotides of each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule toform at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, whereinone or both ends of the siNA molecule are blunt ends. In one embodiment,each of the two 3′ terminal nucleotides of each fragment of the siNAmolecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region, where about 19 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the XIAP gene. In another embodiment, about 21 nucleotides ofthe antisense region are base-paired to the nucleotide sequence or aportion thereof of the RNA encoded by the XIAP gene. In any of the aboveembodiments, the 5′-end of the fragment comprising said antisense regioncan optionally include a phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa XIAP RNA sequence (e.g., wherein said target RNA sequence is encodedby a XIAP gene involved in the XIAP pathway), wherein the siNA moleculedoes not contain any ribonucleotides and wherein each strand of thedouble stranded siNA molecule is about 15 to about 30 nucleotides. Inone embodiment, the siNA molecule is 21 nucleotides in length. Examplesof non-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries shown in Table IV in any combination ofSense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab8/19, Stab 18/19, Stab 7/20, Stab 8/20, or Stab 18/20 (e.g., any siNAhaving Stab 7, 8, 11, 12, 13, 14, 15, 17, 18, 19, or 20 sense orantisense strands or any combination thereof).

In one embodiment, the invention features a chemically synthesizeddouble stranded RNA molecule that directs cleavage of a XIAP RNA via RNAinterference, wherein each strand of said RNA molecule is about 15 toabout 30 nucleotides in length; one strand of the RNA molecule comprisesnucleotide sequence having sufficient complementarity to the XIAP RNAfor the RNA molecule to direct cleavage of the XIAP RNA via RNAinterference; and wherein at least one strand of the RNA moleculeoptionally comprises one or more chemically modified nucleotidesdescribed herein, such as without limitation deoxynucleotides,2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides,2′-O-methoxyethyl nucleotides etc.

In one embodiment, the invention features a medicament comprising ansiNA molecule of the invention.

In one embodiment, the invention features an active ingredientcomprising an siNA molecule of the invention.

In one embodiment, the invention features the use of a double strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a XIAP gene, wherein the siNAmolecule comprises one or more chemical modifications and each strand ofthe double stranded siNA is independently about 15 to about 30 or more(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 or more) nucleotides long. In one embodiment, the siNA molecule ofthe invention is a double stranded nucleic acid molecule comprising oneor more chemical modifications, where each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g. about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of thestrands comprises at least 15 nucleotides that are complementary tonucleotide sequence of XIAP encoding RNA or a portion thereof. In anon-limiting example, each of the two fragments of the siNA moleculecomprise about 21 nucleotides. In another embodiment, the siNA moleculeis a double stranded nucleic acid molecule comprising one or morechemical modifications, where each strand is about 21 nucleotide longand where about 19 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule, wherein at least two 3′ terminal nucleotides of eachfragment of the siNA molecule are not base-paired to the nucleotides ofthe other fragment of the siNA molecule. In another embodiment, the siNAmolecule is a double stranded nucleic acid molecule comprising one ormore chemical modifications, where each strand is about 19 nucleotidelong and where the nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or19) base pairs, wherein one or both ends of the siNA molecule are bluntends. In one embodiment, each of the two 3′ terminal nucleotides of eachfragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, suchas a 2′-deoxy-thymidine. In another embodiment, all nucleotides of eachfragment of the siNA molecule are base-paired to the complementarynucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acid moleculeof about 19 to about 25 base pairs having a sense region and anantisense region and comprising one or more chemical modifications,where about 19 nucleotides of the antisense region are base-paired tothe nucleotide sequence or a portion thereof of the RNA encoded by theXIAP gene. In another embodiment, about 21 nucleotides of the antisenseregion are base-paired to the nucleotide sequence or a portion thereofof the RNA encoded by the XIAP gene. In any of the above embodiments,the 5′-end of the fragment comprising said antisense region canoptionally include a phosphate group.

In one embodiment, the invention features the use of a double strandedshort interfering nucleic acid (siNA) molecule that inhibits,down-regulates, or reduces expression of a XIAP gene, wherein one of thestrands of the double stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of XIAP RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a XIAP gene, wherein one of the strands of thedouble stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence of XIAPRNA or a portion thereof, wherein the other strand is a sense strandwhich comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand and wherein a majority ofthe pyrimidine nucleotides present in the double stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a XIAP gene, wherein one of the strands of thedouble stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence of XIAPRNA that encodes a protein or portion thereof, the other strand is asense strand which comprises nucleotide sequence that is complementaryto a nucleotide sequence of the antisense strand and wherein a majorityof the pyrimidine nucleotides present in the double stranded siNAmolecule comprises a sugar modification. In one embodiment, each strandof the siNA molecule comprises about 15 to about 30 or more (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ormore) nucleotides, wherein each strand comprises at least about 15nucleotides that are complementary to the nucleotides of the otherstrand. In one embodiment, the siNA molecule is assembled from twooligonucleotide fragments, wherein one fragment comprises the nucleotidesequence of the antisense strand of the siNA molecule and a secondfragment comprises nucleotide sequence of the sense region of the siNAmolecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aXIAP gene, wherein a majority of the pyrimidine nucleotides present inthe double stranded siNA molecule comprises a sugar modification, eachof the two strands of the siNA molecule can comprise about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule. In another embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In one embodiment, each strand of the siNA molecule is base-paired tothe complementary nucleotides of the other strand of the siNA molecule.In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of theantisense strand are base-paired to the nucleotide sequence of the XIAPRNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g.,about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of the antisensestrand are base-paired to the nucleotide sequence of the XIAP RNA or aportion thereof.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aXIAP gene, wherein one of the strands of the double stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of XIAP RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double stranded siNA molecule comprises a sugar modification, andwherein the 5′-end of the antisense strand optionally includes aphosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aXIAP gene, wherein one of the strands of the double stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of XIAP RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the untranslatedregion or a portion thereof of the XIAP RNA.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of aXIAP gene, wherein one of the strands of the double stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of XIAP RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand, wherein a majority of the pyrimidine nucleotidespresent in the double stranded siNA molecule comprises a sugarmodification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the XIAP or aportion thereof that is present in the XIAP RNA.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention in a pharmaceutically acceptable carrieror diluent.

In a non-limiting example, the introduction of chemically modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of an siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of an siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding XIAP and thesense region can comprise sequence complementary to the antisenseregion. The siNA molecule can comprise two distinct strands havingcomplementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against XIAP inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising abackbone modified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring or chemicallymodified, each X and Y is independently O, S, N, alkyl, or substitutedalkyl, each Z and W is independently O, S, N, alkyl, substituted alkyl,O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Zare optionally not all O. In another embodiment, a backbone modificationof the invention comprises a phosphonoacetate and/orthiophosphonoacetate internucleotide linkage (see for example Sheehan etal., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, an siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against XIAP inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula II at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotides or non-nucleotides ofFormula II at the 5′-end of the sense strand, the antisense strand, orboth strands. In another non-limiting example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically modified nucleotides ornon-nucleotides of Formula II at the 3′-end of the sense strand, theantisense strand, or both strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against XIAP inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides ornon-nucleotides having Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or more chemicallymodified nucleotide or non-nucleotide of Formula III at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense strand, the antisensestrand, or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) chemically modified nucleotide(s) or non-nucleotide(s) ofFormula III at the 5′-end of the sense strand, the antisense strand, orboth strands. In another non-limiting example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically modified nucleotide ornon-nucleotide of Formula III at the 3′-end of the sense strand, theantisense strand, or both strands.

In another embodiment, an siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against XIAP inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises a 5′-terminalphosphate group having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are not all O.

In one embodiment, the invention features an siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features an siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of an siNA molecule of the invention,for example an siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) against XIAP inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises one or morephosphorothioate internucleotide linkages. For example, in anon-limiting example, the invention features a chemically modified shortinterfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 ormore phosphorothioate internucleotide linkages in one siNA strand. Inyet another embodiment, the invention features a chemically modifiedshort interfering nucleic acid (siNA) individually having about 1, 2, 3,4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages in bothsiNA strands. The phosphorothioate internucleotide linkages can bepresent in one or both oligonucleotide strands of the siNA duplex, forexample in the sense strand, the antisense strand, or both strands. ThesiNA molecules of the invention can comprise one or morephosphorothioate internucleotide linkages at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the sense strand, the antisense strand,or both strands. For example, an exemplary siNA molecule of theinvention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3,4, 5, or more) consecutive phosphorothioate internucleotide linkages atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine phosphorothioate internucleotide linkages inthe sense strand, the antisense strand, or both strands. In yet anothernon-limiting example, an exemplary siNA molecule of the invention cancomprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) purine phosphorothioate internucleotide linkages in the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features an siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features an siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand are chemicallymodified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features an siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features an siNA moleculecomprising 2′-5′ internucleotide linkages. The 2′-5′ internucleotidelinkage(s) can be at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of one or both siNA sequence strands. In addition, the 2′-5′internucleotide linkage(s) can be present at various other positionswithin one or both siNA sequence strands, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more including every internucleotide linkage of apyrimidine nucleotide in one or both strands of the siNA molecule cancomprise a 2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more including every internucleotide linkage of a purinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage.

In another embodiment, a chemically modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, an siNA molecule of the invention comprises a singlestranded hairpin structure, wherein the siNA is about 36 to about 70(e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification comprising a structure having any ofFormulae I-VII or any combination thereof. For example, an exemplarychemically modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, an siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, an siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically modified siNA molecule of the invention comprisesan asymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, an siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, an siNA molecule of the invention comprises at leastone

(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, forexample a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, an siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H. OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R5, R3,R8 or R13 serves as a point of attachment to the siNA molecule of theinvention.

In another embodiment, an siNA molecule of the invention comprises atleast one

(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substitutedpolyalkyl moieties, for example a compound having Formula VII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-SH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double stranded siNA moleculeof the invention or to a single stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g. a moiety having any of Formula V, VI or VII) of theinvention is at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends ofan siNA molecule of the invention. For example, chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) can be present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense strand, the sense strand, or both antisense andsense strands of the siNA molecule. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the 5′-end and 3′-end of the sense strand andthe 3′-end of the antisense strand of a double stranded siNA molecule ofthe invention. In one embodiment, the chemically modified nucleoside ornon-nucleoside (e.g., a moiety having Formula V, VI or VII) is presentat the terminal position of the 5′-end and 3′-end of the sense strandand the 3′-end of the antisense strand of a double stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the two terminal positions of the 5′-end and 3′-endof the sense strand and the 3′-end of the antisense strand of a doublestranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, an siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a3′-3′,3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, an siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, an siNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides),wherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe sense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in thesense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said sense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any (e.g., one or more or all) purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), andwherein any nucleotides comprising a 3′-terminal nucleotide overhangthat are present in said antisense region are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-deoxy purine nucleotides (e.g., wherein allpurine nucleotides are 2′-deoxy purine nucleotides or alternately aplurality of purine nucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region; wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any (e.g., one or more or all) purine nucleotides present inthe antisense region are 2′-O-methyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-O-methyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) against XIAP inside a cell orreconstituted in vitro system comprising a sense region, wherein one ormore pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides), and an antisenseregion, wherein one or more pyrimidine nucleotides present in theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides). The sense region and/or the antisenseregion can have a terminal cap modification, such as any modificationdescribed herein or shown in FIG. 10, that is optionally present at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense and/orantisense sequence. The sense and/or antisense region can optionallyfurther comprise a 3′-terminal nucleotide overhang having about 1 toabout 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. The overhangnucleotides can further comprise one or more (e.g., about 1, 2, 3, 4 ormore) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. Non-limiting examples of these chemicallymodified siNAs are shown in FIGS. 4 and 5 and Tables III and IV herein.In any of these described embodiments, the purine nucleotides present inthe sense region are alternatively 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides) and one or more purine nucleotides present in the antisenseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Also, in any of these embodiments, one or more purine nucleotidespresent in the sense region are alternatively purine ribonucleotides(e.g., wherein all purine nucleotides are purine ribonucleotides oralternately a plurality of purine nucleotides are purineribonucleotides) and any purine nucleotides present in the antisenseregion are 2′-O-methyl purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides).Additionally, in any of these embodiments, one or more purinenucleotides present in the sense region and/or present in the antisenseregion are alternatively selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides (e.g.,wherein all purine nucleotides are selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides or alternately a plurality of purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a Northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabasic moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against XIAP inside a cell or reconstituted in vitrosystem, wherein the chemical modification comprises a conjugatecovalently attached to the chemically modified siNA molecule.Non-limiting examples of conjugates contemplated by the inventioninclude conjugates and ligands described in Vargeese et al., U.S. Ser.No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein inits entirety, including the drawings. In another embodiment, theconjugate is covalently attached to the chemically modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≧2 nucleotides in length, forexample about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. Inanother embodiment, the nucleotide linker can be a nucleic acid aptamer.By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has a sequence that comprises a sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds (e.g.polyethylene glycols such as those having between 2 and 100 ethyleneglycol units). Specific examples include those described by Seela andKaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic Acids Res. 1987,15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991, 113:6324;Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Ma et al.,Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751; Durandet al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al., Nucleosides &Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301;Ono et al., Biochemistry 1991, 30:9914; Arnold et al., InternationalPublication No. WO 89/02439; Usman et al., International Publication No.WO 95/06731; Dudycz et al., International Publication No. WO 95/11910and Ferentz and Verdine, J. Am. Chem. Soc. 1991, 113:4000, all herebyincorporated by reference herein. A “non-nucleotide” further means anygroup or compound that can be incorporated into a nucleic acid chain inthe place of one or more nucleotide units, including either sugar and/orphosphate substitutions, and allows the remaining bases to exhibit theirenzymatic activity. The group or compound can be abasic in that it doesnot contain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, an siNA molecule canbe assembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, an siNA molecule can beassembled from a single oligonculeotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, V, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, an siNA molecule of the invention is a singlestranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single strandedpolynucleotide having complementarity to a target nucleic acid sequence.In another embodiment, the single stranded siNA molecule of theinvention comprises a 5′-terminal phosphate group. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a2′,3′-cyclic phosphate). In another embodiment, the single stranded siNAmolecule of the invention comprises about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In yet another embodiment, the single stranded siNAmolecule of the invention comprises one or more chemically modifiednucleotides or non-nucleotides described herein. For example, all thepositions within the siNA molecule can include chemically modifiednucleotides such as nucleotides having any of Formulae I-VII, or anycombination thereof to the extent that the ability of the siNA moleculeto support RNAi activity in a cell is maintained.

In one embodiment, an siNA molecule of the invention is a singlestranded siNA molecule that mediates RNAi activity in a cell orreconstituted in vitro system comprising a single strandedpolynucleotide having complementarity to a target nucleic acid sequence,wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 10, that is optionally present at the 3′-end and/or the5′-end. The siNA optionally further comprises about 1 to about 4 or more(e.g., about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the3′-end of the siNA molecule, wherein the terminal nucleotides canfurther comprise one or more (e.g., 1, 2, 3, 4 or more)phosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages, and wherein the siNA optionally furthercomprises a terminal phosphate group, such as a 5′-terminal phosphategroup. In any of these embodiments, any purine nucleotides present inthe antisense region are alternatively 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA (i.e., purine nucleotides present in the senseand/or antisense region) can alternatively be locked nucleic acid (LNA)nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides oralternately a plurality of purine nucleotides are LNA nucleotides).Also, in any of these embodiments, any purine nucleotides present in thesiNA are alternatively 2′-methoxyethyl purine nucleotides (e.g., whereinall purine nucleotides are 2′-methoxyethyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides). In another embodiment, any modified nucleotides present inthe single stranded siNA molecules of the invention comprise modifiednucleotides having properties or characteristics similar to naturallyoccurring ribonucleotides. For example, the invention features siNAmolecules including modified nucleotides having a Northern conformation(e.g., Northern pseudorotation cycle, see for example Saenger,Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Assuch, chemically modified nucleotides present in the single strandedsiNA molecules of the invention are preferably resistant to nucleasedegradation while at the same time maintaining the capacity to mediateRNAi.

In one embodiment, an siNA molecule of the invention compriseschemically modified nucleotides or non-nucleotides (e.g., having any ofFormulae I-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides) at alternating positions within one or more strands orregions of the siNA molecule. For example, such chemical modificationscan be introduced at every other position of a RNA based siNA molecule,starting at either the first or second nucleotide from the 3′-end or5′-end of the siNA. In a non-limiting example, a double stranded siNAmolecule of the invention in which each strand of the siNA is 21nucleotides in length is featured wherein positions 1, 3, 5, 7, 9, 11,13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., withcompounds having any of Formulae I-VII, such as such as 2′-deoxy,2′-deoxy-2′-fluoro, or 2′-O-methyl nucleotides). In another non-limitingexample, a double stranded siNA molecule of the invention in which eachstrand of the siNA is 21 nucleotides in length is featured whereinpositions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand arechemically modified (e.g., with compounds having any of Formulae I-VII,such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, or 2′-O-methylnucleotides). Such siNA molecules can further comprise terminal capmoieties and/or backbone modifications as described herein.

In one embodiment, the invention features a method for modulating theexpression of a XIAP gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the XIAP gene; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the XIAP gene inthe cell.

In one embodiment, the invention features a method for modulating theexpression of a XIAP gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the XIAP gene and wherein the sense strand sequence of the siNAcomprises a sequence identical or substantially similar to the sequenceof the target RNA; and (b) introducing the siNA molecule into a cellunder conditions suitable to modulate the expression of the XIAP gene inthe cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one XIAP gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the XIAP genes; and (b) introducing the siNAmolecules into a cell under conditions suitable to modulate theexpression of the XIAP genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more XIAP genes within a cell comprising: (a)synthesizing one or more siNA molecules of the invention, which can bechemically modified, wherein the siNA strands comprise sequencescomplementary to RNA of the XIAP genes and wherein the sense strandsequences of the siNAs comprise sequences identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecules into a cell under conditions suitable to modulate theexpression of the XIAP genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one XIAP gene within a cell comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the XIAP gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequences of the target RNAs; and (b) introducing thesiNA molecule into a cell under conditions suitable to modulate theexpression of the XIAP genes in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. In one embodiment, theinvention features a method of modulating the expression of a XIAP genein a tissue explant comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands comprises a sequence complementary to RNA of the XIAP gene; and(b) introducing the siNA molecule into a cell of the tissue explantderived from a particular organism under conditions suitable to modulatethe expression of the XIAP gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of theXIAP gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a XIAP gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the XIAP gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequence of the target RNA; and (b) introducing the siNAmolecule into a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of theXIAP gene in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the XIAP gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one XIAP gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the XIAP genes; and (b) introducing thesiNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the XIAP genes in the tissue explant. In another embodiment, themethod further comprises introducing the tissue explant back into theorganism the tissue was derived from or into another organism underconditions suitable to modulate the expression of the XIAP genes in thatorganism.

In one embodiment, the invention features a method of modulating theexpression of a XIAP gene in a subject or organism comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the XIAP gene; and (b) introducing the siNAmolecule into the subject or organism under conditions suitable tomodulate the expression of the XIAP gene in the subject or organism. Thelevel of XIAP protein or RNA can be determined using various methodswell-known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one XIAP gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically modified, wherein one of the siNA strands comprises asequence complementary to RNA of the XIAP genes; and (b) introducing thesiNA molecules into the subject or organism under conditions suitable tomodulate the expression of the XIAP genes in the subject or organism.The level of XIAP protein or RNA can be determined as is known in theart.

In one embodiment, the invention features a method for modulating theexpression of a XIAP gene within a cell comprising: (a) synthesizing ansiNA molecule of the invention, which can be chemically modified,wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the XIAP gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate theexpression of the XIAP gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one XIAP gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can be chemicallymodified, wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the XIAP gene; and (b) contacting the cell invitro or in vivo with the siNA molecule under conditions suitable tomodulate the expression of the XIAP genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a XIAP gene in a tissue explant comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the XIAP gene; and (b) contacting a cell ofthe tissue explant derived from a particular subject or organism withthe siNA molecule under conditions suitable to modulate the expressionof the XIAP gene in the tissue explant. In another embodiment, themethod further comprises introducing the tissue explant back into thesubject or organism the tissue was derived from or into another subjector organism under conditions suitable to modulate the expression of theXIAP gene in that subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one XIAP gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the XIAP gene; and (b)introducing the siNA molecules into a cell of the tissue explant derivedfrom a particular subject or organism under conditions suitable tomodulate the expression of the XIAP genes in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the subject or organism the tissue was derived from orinto another subject or organism under conditions suitable to modulatethe expression of the XIAP genes in that subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a XIAP gene in a subject or organism comprising: (a)synthesizing an siNA molecule of the invention, which can be chemicallymodified, wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the XIAP gene; and (b) introducing the siNAmolecule into the subject or organism under conditions suitable tomodulate the expression of the XIAP gene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one XIAP gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the XIAP gene; and (b)introducing the siNA molecules into the subject or organism underconditions suitable to modulate the expression of the XIAP genes in thesubject or organism.

In one embodiment, the invention features a method of modulating theexpression of a XIAP gene in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the XIAP gene in thesubject or organism.

In one embodiment, the invention features a method for treating orpreventing cancer, such as ovarian cancer; cancers of non-lymphoidparenchymal organs including the heart, placenta, skeletal muscle andlung; breast cancer; cancers of the head and neck, including variouslymphomas such as mantle cell lymphoma; non-Hodgkins lymphoma; adenoma;squamous cell carcinoma; laryngeal carcinoma; cancers of the retina;cancers of the esophagus; multiple myeloma; melanoma; colorectal cancer;lung cancer; bladder cancer; prostate cancer; and glioblastomacomprising contacting the subject or organism with an siNA molecule ofthe invention under conditions suitable to modulate the expression ofXIAP gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing proliferative disorders, such as restenosis, comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of theXIAP gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing polycystic kidney disease in a subject or organism comprisingcontacting the subject or organism with an siNA molecule of theinvention under conditions suitable to modulate the expression of theXIAP gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing ocular disease in a subject or organism comprising contactingthe subject or organism with an siNA molecule of the invention underconditions suitable to modulate the expression of the XIAP gene in thesubject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one XIAP genes in a subject or organismcomprising contacting the subject or organism with one or more siNAmolecules of the invention under conditions suitable to modulate theexpression of the XIAP genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate orinhibit target (e.g., XIAP) gene expression through RNAi targeting of avariety of RNA molecules. In one embodiment, the siNA molecules of theinvention are used to target various RNAs corresponding to a targetgene. Non-limiting examples of such RNAs include messenger RNA (mRNA),alternate RNA splice variants of target gene(s), post-transcriptionallymodified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNAtemplates. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as XIAP family genes. As such, siNA molecules targetingmultiple XIAP targets can provide increased therapeutic effect. Inaddition, siNA can be used to characterize pathways of gene function ina variety of applications. For example, the present invention can beused to inhibit the activity of target gene(s) in a pathway to determinethe function of uncharacterized gene(s) in gene function analysis, mRNAfunction analysis, or translational analysis. The invention can be usedto determine potential target gene pathways involved in various diseasesand conditions toward pharmaceutical development. The invention can beused to understand pathways of gene expression involved in, for example,cancer and other proliferative disorders.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession Nos., for example, XIAP genes encoding RNAsequence(s) referred to herein by Genbank Accession number, for example,Genbank Accession Nos. shown in Table I.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described herein. In another embodiment, the assaycan comprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of target RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, Northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4N, where N represents the numberof base paired nucleotides in each of the siNA construct strands (e.g.,for an siNA construct having 21 nucleotide sense and antisense strandswith 19 base pairs, the complexity would be 4¹⁹); and (b) assaying thesiNA constructs of (a) above, under conditions suitable to determineRNAi target sites within the target XIAP RNA sequence. In anotherembodiment, the siNA molecules of (a) have strands of a fixed length,for example about 23 nucleotides in length. In yet another embodiment,the siNA molecules of (a) are of differing length, for example havingstrands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In oneembodiment, the assay can comprise a reconstituted in vitro siNA assayas described in Example 6 herein. In another embodiment, the assay cancomprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of XIAP RNA are analyzed for detectablelevels of cleavage, for example, by gel electrophoresis, Northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target XIAP RNA sequence. The target XIAP RNAsequence can be obtained as is known in the art, for example, by cloningand/or transcription for in vitro systems, and by cellular expression inin vivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length.

In another embodiment, the siNA molecules of (b) are of differinglength, for example having strands of about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides in length. In one embodiment, the assay can comprise areconstituted in vitro siNA assay as described herein. In anotherembodiment, the assay can comprise a cell culture system in which targetRNA is expressed. Fragments of target RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, Northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by expression in in vivosystems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by an siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising ansiNA molecule of the invention, which can be chemically modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds.

In another embodiment, the invention features a method for validating aXIAP gene target, comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a XIAP target gene;(b) introducing the siNA molecule into a cell, tissue, subject, ororganism under conditions suitable for modulating expression of the XIAPtarget gene in the cell, tissue, subject, or organism; and (c)determining the function of the gene by assaying for any phenotypicchange in the cell, tissue, subject, or organism.

In another embodiment, the invention features a method for validating aXIAP target comprising: (a) synthesizing an siNA molecule of theinvention, which can be chemically modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a XIAP target gene;(b) introducing the siNA molecule into a biological system underconditions suitable for modulating expression of the XIAP target gene inthe biological system; and (c) determining the function of the gene byassaying for any phenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, subject, or organism, or extract thereof. The term biologicalsystem also includes reconstituted RNAi systems that can be used in anin vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing an siNAmolecule of the invention, which can be chemically modified, that can beused to modulate the expression of a XIAP target gene in a biologicalsystem, including, for example, in a cell, tissue, subject, or organism.In another embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically modified,that can be used to modulate the expression of more than one XIAP targetgene in a biological system, including, for example, in a cell, tissue,subject, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically modified. Inanother embodiment, the cell containing an siNA molecule of theinvention is a mammalian cell. In yet another embodiment, the cellcontaining an siNA molecule of the invention is a human cell.

In one embodiment, the synthesis of an siNA molecule of the invention,which can be chemically modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing ansiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double stranded siNA molecule.

In another embodiment, the invention features a method for synthesizingan siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating the doublestranded siNA molecule, for example using a trityl-on synthesis strategyas described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against XIAP, wherein the siNA construct comprises one or morechemical modifications, for example, one or more chemical modificationshaving any of Formulae I-VII or any combination thereof that increasesthe nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In another embodiment, the invention features a method for generatingsiNA molecules with improved toxicologic profiles (e.g., have attenuatedor no immunostimulatory properties) comprising (a) introducingnucleotides having any of Formula I-VII (e.g. siNA motifs referred to inTable IV) or any combination thereof into an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) introducing nucleotides having anyof Formula I-VII (e.g. siNA motifs referred to in Table IV) or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate an interferon response.

By “improved toxicological profile”, is meant, that the chemicallymodified siNA construct exhibits decreased toxicity in a cell, subject,or organism compared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In a non-limiting example, siNA molecules withimproved toxicologic profiles are associated with a decreased orattenuated immunostimulatory response in a cell, subject, or organismcompared to an unmodified siNA or siNA molecule having fewermodifications or modifications that are less effective in impartingimproved toxicology. In one embodiment, an siNA molecule with animproved toxicological profile comprises no ribonucleotides. In oneembodiment, an siNA molecule with an improved toxicological profilecomprises less than 5 ribonucleotides (e.g., 1, 2, 3, or 4ribonucleotides). In one embodiment, an siNA molecule with an improvedtoxicological profile comprises Stab 7, Stab 8, Stab 11, Stab 12, Stab13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24, Stab25, Stab 26, Stab 27, Stab 28 or any combination thereof (see Table IV).In one embodiment, the level of immunostimulatory response associatedwith a given siNA molecule can be measured as is known in the art, forexample by determining the level of PKR/interferon response,proliferation, B-cell activation, and/or cytokine production in assaysto quantitate the immunostimulatory response of particular siNAmolecules (see for example Leifer et al., 2003, J. Immunother. 26, 313-9and U.S. Pat. No. 5,968,909, incorporated by reference herein).

In one embodiment, the invention features siNA constructs that mediateRNAi against XIAP, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoan siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against XIAP, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against XIAP, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the bindingaffinity between the antisense strand of the siNA construct and acomplementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against XIAP, wherein the siNA construct comprises one or morechemical modifications described herein that modulate the polymeraseactivity of a cellular polymerase capable of generating additionalendogenous siNA molecules having sequence homology to the chemicallymodified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically modified siNAmolecule.

In one embodiment, the invention features chemically modified siNAconstructs that mediate RNAi against XIAP in a cell, wherein thechemical modifications do not significantly effect the interaction ofsiNA with a target RNA molecule, DNA molecule and/or proteins or otherfactors that are essential for RNAi in a manner that would decrease theefficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against XIAP comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against XIAPtarget RNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against XIAPtarget DNA comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into an siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against XIAP, wherein the siNA construct comprises one or morechemical modifications described herein that modulates the cellularuptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against XIAP with improved cellular uptake comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into an siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating siNA molecules havingimproved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against XIAP, wherein the siNA construct comprises one or morechemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of an siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, an siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free. 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, “Stab 24/25” and “Stab 24/26” (e.g., any siNA having Stab7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (seeTable IV) wherein the 5′-end and 3′-end of the sense strand of the siNAdo not comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of an siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”,“Stab 24/25” and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23,or 24 sense strands) chemistries and variants thereof (see Table IV)wherein the 5′-end and 3′-end of the sense strand of the siNA do notcomprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g. chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to an siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into an siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include an siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 4-6, and Tables IIand III herein. For example the siNA can be a double strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof (e.g., about 15 to about 25 or morenucleotides of the siNA molecule are complementary to the target nucleicacid or a portion thereof). Alternatively, the siNA is assembled from asingle oligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der Waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide, chemicallymodified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), andothers. In addition, as used herein, the term RNAi is meant to beequivalent to other terms used to describe sequence specific RNAinterference, such as post transcriptional gene silencing, translationalinhibition, or epigenetics. For example, siNA molecules of the inventioncan be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation pattern to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237).

In one embodiment, an siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. US04/16390, filed May 24, 2004).

In one embodiment, an siNA molecule of the invention is amultifunctional siNA, (see for example FIGS. 16-21 and Jadhav et al.,U.S. Ser. No. 60/543,480 filed Feb. 10, 2004 and International PCTApplication No. US04/16390, filed May 24, 2004). The multifunctionalsiNA of the invention can comprise sequence targeting, for example, tworegions of XIAP RNA (see for example target sequences in Tables II andIII).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example; an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant an siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g. RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing.

By “gene”, or “target gene”, is meant a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Aberrant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of asubject, organism or cell, by intervening in cellular processes such asgenetic imprinting, transcription, translation, or nucleic acidprocessing (e.g., transamination, methylation etc.). The target gene canbe a gene derived from a cell, an endogenous gene, a transgene, orexogenous genes such as genes of a pathogen, for example a virus, whichis present in the cell after infection thereof. The cell containing thetarget gene can be derived from or contained in any organism, forexample a plant, animal, protozoan, virus, bacterium, or fungus.Non-limiting examples of plants include monocots, dicots, orgymnosperms. Non-limiting examples of animals include vertebrates orinvertebrates. Non-limiting examples of fungi include molds or yeasts.For a review, see for example Snyder and Gerstein, 2003, Science, 300,258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-C5-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “XIAP” as used herein is meant, any X-linked inhibitor of apoptosis(XIAP) protein, peptide, or polypeptide having XIAP activity, such asencoded by XIAP Genbank Accession Nos. shown in Table I. The term XIAPalso refers to nucleic acid sequences encoding any XIAP protein,peptide, or polypeptide having XIAP activity. The term XIAP as usedherein also refers to other inhibitor of apoptosis genes (XIAP) encodinginhibitor of apoptosis proteins, such as HIAP1, HIAP2, and/or NAIP. Theterm “XIAP” is also meant to include other XIAP encoding sequence, suchas XIAP isoforms, mutant XIAP genes, splice variants of XIAP genes, andXIAP gene polymorphisms. By “homologous sequence” is meant, a nucleotidesequence that is shared by one or more polynucleotide sequences, such asgenes, gene transcripts and/or non-coding polynucleotides. For example,a homologous sequence can be a nucleotide sequence that is shared by twoor more genes encoding related but different proteins, such as differentmembers of a gene family, different protein epitopes, different proteinisoforms or completely divergent genes, such as a cytokine and itscorresponding receptors. A homologous sequence can be a nucleotidesequence that is shared by two or more non-coding polynucleotides, suchas noncoding DNA or RNA, regulatory sequences, introns, and sites oftranscriptional control or regulation. Homologous sequences can alsoinclude conserved sequence regions shared by more than onepolynucleotide sequence. Homology does not need to be perfect homology(e.g., 100%), as partially homologous sequences are also contemplated bythe instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of an siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of an siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of an siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence. In one embodiment, an siNA moleculeof the invention comprises about 15 to about 30 or more (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)nucleotides that are complementary to one or more target nucleic acidmolecules or a portion thereof.

In one embodiment, siNA molecules of the invention that down regulate orreduce XIAP gene expression are used for preventing or treating, in asubject or organism, a variety of oncogenic and proliferative diseasesand disorders. By “proliferative disease” or “cancer” as used herein ismeant, any disease or condition characterized by unregulated cell growthor replication as is known in the art, including various cancersincluding but not limited to multiple drug resistant cancers, such asleukemias including acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), Acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia; AIDS related cancers such as Kaposi'ssarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression (e.g., XIAP) in a cell ortissue, alone or in combination with other therapies.

By “ocular disease” as used herein is meant, any disease, condition,trait, genotype or phenotype of the eye and related structures, such asCystoid Macular Edema, Asteroid Hyalosis, Pathological Myopia andPosterior Staphyloma, Toxocariasis (Ocular Larva Migrans), Retinal VeinOcclusion, Posterior Vitreous Detachment, Tractional Retinal Tears,Epiretinal Membrane, Diabetic Retinopathy, Lattice Degeneration, RetinalVein Occlusion, Retinal Artery Occlusion, Macular Degeneration (e.g.,age related macular degeneration such as wet AMD or dry AMD),Toxoplasmosis, Choroidal Melanoma, Acquired Retinoschisis, HollenhorstPlaque, Idiopathic Central Serous Chorioretinopathy, Macular Hole,Presumed Ocular Histoplasmosis. Syndrome, Retinal Macroaneursym,Retinitis Pigmentosa, Retinal Detachment, Hypertensive Retinopathy,Retinal Pigment Epithelium (RPE) Detachment, Papillophlebitis, OcularIschemic Syndrome, Coats' Disease, Leber's Miliary Aneurysm,Conjunctival Neoplasms, Allergic Conjunctivitis, Vernal Conjunctivitis,Acute Bacterial Conjunctivitis, Allergic Conjunctivitis &VernalKeratoconjunctivitis, Viral Conjunctivitis, Bacterial Conjunctivitis,Chlamydial & Gonococcal Conjunctivitis, Conjunctival Laceration,Episcleritis, Scleritis, Pingueculitis, Pterygium, Superior LimbicKeratoconjunctivitis (SLK of Theodore), Toxic Conjunctivitis,Conjunctivitis with Pseudomembrane, Giant Papillary Conjunctivitis,Terrien's Marginal Degeneration, Acanthamoeba Keratitis, FungalKeratitis, Filamentary Keratitis, Bacterial Keratitis, KeratitisSicca/Dry Eye Syndrome, Bacterial Keratitis, Herpes Simplex Keratitis,Sterile Corneal Infiltrates, Phlyctenulosis, Corneal Abrasion &Recurrent Corneal Erosion, Corneal Foreign Body, Chemical Burs,Epithelial Basement Membrane Dystrophy (EBMD), Thygeson's SuperficialPunctate Keratopathy, Corneal Laceration, Salzmann's NodularDegeneration, Fuchs' Endothelial Dystrophy, Crystalline LensSubluxation, Ciliary-Block Glaucoma, Primary Open-Angle Glaucoma,Pigment Dispersion Syndrome and Pigmentary Glaucoma, PseudoexfoliationSyndrome and Pseudoexfoliative Glaucoma, Anterior Uveitis, Primary OpenAngle Glaucoma, Uveitic Glaucoma & Glaucomatocyclitic Crisis, PigmentDispersion Syndrome & Pigmentary Glaucoma, Acute Angle Closure Glaucoma,Anterior Uveitis, Hyphema, Angle Recession Glaucoma, Lens InducedGlaucoma, Pseudoexfoliation Syndrome and Pseudoexfoliative Glaucoma,Axenfeld-Rieger Syndrome, Neovascular Glaucoma, Pars Planitis, ChoroidalRupture, Duane's Retraction Syndrome, Toxic/Nutritional OpticNeuropathy, Aberrant Regeneration of Cranial Nerve III, IntracranialMass Lesions, Carotid-Cavernous Sinus Fistula, Anterior Ischemic OpticNeuropathy, Optic Disc Edema & Papilledema, Cranial Nerve III Palsy,Cranial Nerve IV Palsy, Cranial Nerve VI Palsy, Cranial Nerve VII(Facial Nerve) Palsy, Horner's Syndrome, Internuclear Opthalmoplegia,Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil, Optic Nerve HeadDrusen, Demyelinating Optic Neuropathy (Optic Neuritis, RetrobulbarOptic Neuritis), Amaurosis Fugax and Transient Ischemic Attack,Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal CellCarcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis,Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes SimplexBlepharitis, Orbital Cellulitis, Senile Entropion, and Squamous CellCarcinoma.

In one embodiment, the siNA molecules of the invention are used to treator prevent cancer or other proliferative disorders in a subject ororganism.

In one embodiment, the siNA molecules of the invention are used to treator prevent ocular disease in a subject or organism.

In one embodiment of the present invention, each sequence of an siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs. Exemplary siNA molecules of the invention are shown in TableII. Exemplary synthetic siNA molecules of the invention are shown inTable III and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through direct dermal application, transdermal application, orinjection, with or without their incorporation in biopolymers. Inparticular embodiments, the nucleic acid molecules of the inventioncomprise sequences shown in Tables II-III and/or FIGS. 4-5. Examples ofsuch nucleic acid molecules consist essentially of sequences defined inthese tables and figures. Furthermore, the chemically modifiedconstructs described in Table IV can be applied to any siNA sequence ofthe invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribofuranose moiety. The terms includedouble stranded RNA, single stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating cancer or other proliferative disorder in asubject or organism.

For example, the siNA molecules can be administered to a subject or canbe administered to other appropriate cells evident to those skilled inthe art, individually or in combination with one or more drugs underconditions suitable for the treatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to prevent or treat cancer or otherproliferative disorder in a subject or organism. For example, thedescribed molecules could be used in combination with one or more knowncompounds, treatments, or procedures to prevent or treat cancer or otherproliferative disorder in a subject or organism as are known in the art.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of an siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms an siNA molecule. Non-limitingexamples of such expression vectors are described in Paul et al., 2002,Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, NatureBiotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500;and Novina et al., 2002, Nature Medicine, advance online publicationdoi: 10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for an siNA molecule having complementarity to aRNA molecule referred to by a Genbank Accession numbers, for exampleGenbank Accession Nos. shown in Table I.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand I andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form ansiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4A-F shows non-limiting examples of chemically modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs. Theantisense strand of constructs A-F comprise sequence complementary toany target nucleic acid sequence of the invention. Furthermore, when aglyceryl moiety (L) is present at the 3′-end of the antisense strand forany construct shown in FIG. 4 A-F, the modified internucleotide linkageis optional.

FIG. 4A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s”, optionally connects the (N N)nucleotides in the sense and antisense strand.

FIG. 4C: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. The antisense strand comprises 21 nucleotides,optionally having a 3′-terminal glyceryl moiety and wherein the twoterminal 3′-nucleotides are optionally complementary to the target RNAsequence, and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4D: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

FIG. 4E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

FIG. 4F: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

FIG. 5A-F shows non-limiting examples of specific chemically modifiedsiNA sequences of the invention. A-F applies the chemical modificationsdescribed in FIG. 4A-F to a XIAP siNA sequence. Such chemicalmodifications can be applied to any XIAP sequence and/or XIAPpolymorphism sequence.

FIG. 6 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct I invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIG. 7A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

FIG. 7A: A DNA oligomer is synthesized with a 5′-restriction site (R1)sequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined XIAP target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, which is followed by a loop sequence of defined sequence (X),comprising, for example, about 3 to about 10 nucleotides.

FIG. 7B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence thatwill result in an siNA transcript having specificity for a XIAP targetsequence and having self-complementary sense and antisense regions.

FIG. 7C: The construct is heated (for example to about 95° C.) tolinearize the sequence, thus allowing extension of a complementarysecond DNA strand using a primer to the 3′-restriction sequence of thefirst strand. The double stranded DNA is then inserted into anappropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8A-C is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double stranded siNAconstructs.

FIG. 8A: A DNA oligomer is synthesized with a 5′-restriction (R1) sitesequence followed by a region having sequence identical (sense region ofsiNA) to a predetermined XIAP target sequence, wherein the sense regioncomprises, for example, about 19, 20, 21, or 22 nucleotides (N) inlength, and which is followed by a 3′-restriction site (R2) which isadjacent to a loop sequence of defined sequence (X).

FIG. 8B: The synthetic construct is then extended by DNA polymerase togenerate a hairpin structure having self-complementary sequence.

FIG. 8C: The construct is processed by restriction enzymes specific toR1 and R2 to generate a double stranded DNA which is then inserted intoan appropriate vector for expression in cells. The transcriptioncassette is designed such that a U6 promoter region flanks each side ofthe dsDNA which generates the separate sense and antisense strands ofthe siNA. Poly T termination sequences can be added to the constructs togenerate U overhangs in the resulting transcript.

FIG. 9A-E is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

FIG. 9A: A pool of siNA oligonucleotides are synthesized wherein theantisense region of the siNA constructs has complementarity to targetsites across the target nucleic acid sequence, and wherein the senseregion comprises sequence complementary to the antisense region of thesiNA.

FIGS. 9B&C: (FIG. 9B) The sequences are pooled and are inserted intovectors such that (FIG. 9C) transfection of a vector into cells resultsin the expression of the siNA.

FIG. 9D: Cells are sorted based on phenotypic change that is associatedwith modulation of the target nucleic acid sequence.

FIG. 9E: The siNA is isolated from the sorted cells and is sequenced toidentify efficacious target sites within the target nucleic acidsequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8). [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g. humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palidrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palidrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. FIG. 16A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 16B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first and second complementaryregions are situated at the 5′-ends of each polynucleotide sequence inthe multifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. FIG. 17A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. FIG. 17B shows a non-limiting example of a multifunctionalsiNA molecule having a first region that is complementary to a firsttarget nucleic acid sequence (complementary region 1) and a secondregion that is complementary to a second target nucleic acid sequence(complementary region 2), wherein the first complementary region issituated at the 5′-end of the polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. In one embodiment,these multifunctional siNA constructs are processed in vivo or in vitroto generate multifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifuctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. FIG. 18Ashows a non-limiting example of a multifunctional siNA molecule having afirst region that is complementary to a first target nucleic acidsequence (complementary region 1) and a second region that iscomplementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 3′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 18B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 5′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifuctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. FIG. 19A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. FIG. 19B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins,for example, a cytokine and its corresponding receptor, differing viralstrains, a virus and a cellular protein involved in viral infection orreplication, or differing proteins involved in a common or divergentbiologic pathway that is implicated in the maintenance of progression ofdisease. Each strand of the multifunctional siNA construct comprises aregion having complementarity to separate target nucleic acid molecules.The multifunctional siNA molecule is designed such that each strand ofthe siNA can be utilized by the RISC complex to initiate RNAinterference mediated cleavage of its corresponding target. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarityto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 22 shows a non-limiting example of reduction of XIAP mRNA in A549cells mediated by chemically modified siNAs that target XIAP mRNA. A549cells were transfected with 0.25 ug/well of lipid complexed with 25 nMsiNA. Active siNA constructs comprising various stabilizationchemistries (see Tables III and IV) were compared to untreated cells,matched chemistry irrelevant siNA control constructs (IC1, IC2), andcells transfected with lipid alone (transfection control). As shown inthe figure, the siNA constructs significantly reduce XIAP RNAexpression.

DETAILED DESCRIPTION OF THE INVENTION Mechanism of Action of NucleicAcid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or an siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′,5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing an siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of an siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et'al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 mmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM 12, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1,2-benzodithiol-3-one1, 1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1,2-benzodithiol-3-one 1,1-dioxide, 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA·3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA·3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995Nucleic Acids. Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

An siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al, supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat; U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to an siNA molecule of the invention or thesense and antisense strands of an siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologicaily activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect an siNA molecule of the invention comprises one ormore 5′ and/or a 3′-cap structure, for example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and suitable heterocyclic groups include furanyl, thienyl, pyridyl,pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl, pyrazinyl, imidazolyl andthe like, all optionally substituted. An “amide” refers to an—C(O)—NH—R, where R is either alkyl, aryl, alkylaryl or hydrogen. An“ester” refers to an —C(O)—OR′, where R is either alkyl, aryl, alkylarylor hydrogen.

“Nucleotide” as used herein, and as recognized in the art, includesnatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

An siNA molecule of the invention can be adapted for use to prevent ortreat cancer or other proliferative disorders and conditions, oculardisease, or any other trait, disease or condition that is related to orwill respond to the levels of XIAP in a cell or tissue, alone or incombination with other therapies. For example, an siNA molecule cancomprise a delivery vehicle, including liposomes, for administration toa subject, carriers and diluents and their salts, and/or can be presentin pharmaceutically acceptable formulations. Methods for the delivery ofnucleic acid molecules are described in Akhtar et al., 1992, Trends CellBio., 2, 139; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol.,16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137,165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all ofwhich are incorporated herein by reference. Beigelman et al., U.S. Pat.No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe thegeneral methods for delivery of nucleic acid molecules. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in United StatesPatent Application Publication No. 20030077829, incorporated byreference herein in its entirety.

In one embodiment, an siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, an siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine anddioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) CytofectinGSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (GlenResearch); (3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PharmaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, an siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. No. 6,528,631; U.S. Pat. No. 6,335,434; U.S. Pat. No. 6,235,886;U.S. Pat. No. 6,153,737; U.S. Pat. No. 5,214,136; U.S. Pat. No.5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as creams, gels, sprays, oilsand other suitable compositions for topical, dermal, or transdermaladministration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdermal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, intraperitoneal, inhalation, oral,intrapulmonary and intramuscular. Each of these administration routesexposes the siNA molecules of the invention to an accessible diseasedtissue. The rate of entry of a drug into the circulation has been shownto be a function of molecular weight or size. The use of a liposome orother drug carrier comprising the compounds of the instant invention canpotentially localize the drug, for example, in certain tissue types,such as the tissues of the reticular endothelial system (RES). Aliposome formulation that can facilitate the association of drug withthe surface of cells, such as, lymphocytes and macrophages is alsouseful. This approach can provide enhanced delivery of the drug totarget cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, D F et al, 1999, Cell Transplant,8, 47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit., 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatennary or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002. Alternatively, certain siNAmolecules of the instant invention can be expressed within cells fromeukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229,345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399;Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591-5;Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic etal., 1992, J. Virol., 66, 1432-41; Weerasinghe et al., 1991, J. Virol.,65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA, 89,10802-6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver etal., 1990 Science, 247, 1222-1225; Thompson et al., 1995, Nucleic AcidsRes., 23, 2259; Good et al., 1997, Gene Therapy, 4, 45. Those skilled inthe art realize that any nucleic acid can be expressed in eukaryoticcells from the appropriate DNA/RNA vector. The activity of such nucleicacids can be augmented by their release from the primary transcript by aenzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan etal., PCT WO 94/02595; Ohkawa et al., 1992, Nucleic Acids Symp. Ser., 27,15-6; Taira et al., 1991, Nucleic Acids Res., 19, 5125-30; Ventura etal., 1993, Nucleic Acids Res., 21, 3249-55; Chowrira et al., 1994, J.Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pats.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systemic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture et al., 1996, TIG.,12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of ansiNA duplex, or a single self-complementary strand that self hybridizesinto an siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi: 10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. U S A, 87, 6743-7;Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al.,1993, Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol.,10, 4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO J., 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U.S. A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, Gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of an siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of an siNA molecule, wherein the sequenceis operably linked to the 3′-end of the open reading frame and whereinthe sequence is operably linked to the initiation region, the intron,the open reading frame and the termination region in a manner whichallows expression and/or delivery of the siNA molecule.

XIAP Biology and Biochemistry

Apoptosis is a physiological cell death process that is important in thedevelopment, homeostasis, and immune defense of multicellular animals.The inhibitor of apoptosis (IAP) gene family encodes a group ofstructurally related proteins that have the ability to suppressapoptotic cell death by binding to and inhibiting caspases (Lotocki etal., 2002, IUBMB Life, 54(5), 231 and Salvesen et al., 2002, NatureReviews Molecular Cell Biology, 3, 401). Caspses are cysteine proteaseswith a substrate preference for aspartic acid and are the key effectorsof apoptosis (Verhagen et al., 2001 GenomeBiology, 2). All IAP's are BIR(baculovirus IAP repeat) containing proteins and BIRs are essential forthe anti-apoptotic properties of the IAP's because they have beenattributed to the binding and inhibition of caspases (Salvesen et al.,supra). IAP's can be induced by the transcription factor NF-KB or v-Rel,and HIAP1 and HIAP2 can activiate NF-KB (LaCasse et al., 1998, Oncogene,17(25), 3247).

XIAP (X-linked inhibitor of apoptosis protein) is a 57-kDA protein(Salvesen et al., supra). XIAP is also a mammalian inhibitor ofapoptosis protein and is a suppressor of apoptotic cell death. XIAPblocks the mitochondrial death pathway by binding directly to certaininitiator and effector caspases. (Li et al., 2003 Hebei Daxue Xuebao,Ziran Kexueban 23, 100). However, XIAP mutants that cannot bind caspasescan still inhibit apoptosis (Salvesen et al., supra). When cells areinfected by a virus, such as cancer, XIAP inhibits the apoptosis thatwould occur and the cancer cells continue inappropriate proliferation.Other IAPs (inhibitor of apoptosis proteins) including HIAP1, HIAP2(human inhibitor or apoptosis 1 and 2), and NAIP (neuronal apoptosisinhibitor protein) can also suppress apoptosis.

Because XIAP and other IAP's, including HIAP1, HIAP2, and NAIP, areinhibitors of apoptosis, modulation of IAP gene expression using RNAinterference medicated by short interfering nucleic acids represents anovel treatment approach for cancer and other proliferative diseases andconditions where the regulation of apoptosis is lost.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1 Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of an siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexafluororophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H₂O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H₂O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H₂O followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2 Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3 Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables II and III). Ifterminal TT residues are desired for the sequence (as described inparagraph 7), then the two 3′ terminal nucleotides of both the sense andantisense strands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see, for example, Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004, doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32, doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a XIAPtarget sequence is used to screen for target sites in cells expressingXIAP RNA, such as cultured human T cells. The general strategy used inthis approach is shown in FIG. 9. A non-limiting example of such is apool comprising sequences having any of SEQ ID NOS 1-1056. Cellsexpressing XIAP (e.g., cultured human T cells) are transfected with thepool of siNA constructs and cells that demonstrate a phenotypeassociated with XIAP inhibition are sorted. The pool of siNA constructscan be expressed from transcription cassettes inserted into appropriatevectors (see for example FIG. 7 and FIG. 8). The siNA from cellsdemonstrating a positive phenotypic change (e.g., decreasedproliferation, decreased XIAP mRNA levels or decreased XIAP proteinexpression), are sequenced to determine the most suitable target site(s)within the target XIAP RNA sequence.

Example 4 XIAP Targeted siNA Design

siNA target sites were chosen by analyzing sequences of the XIAP RNAtarget and optionally prioritizing the target sites on the basis offolding (structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.11).

Example 5 Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphosphoroamidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately; 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. No. 5,831,071, U.S. Pat.No. 6,353,098, U.S. Pat. No. 6,437,117, and Bellon et al., U.S. Pat. No.6,054,576, U.S. Pat. No. 6,162,909, U.S. Pat. No. 6,303,773, or Scaringesupra, incorporated by reference herein in their entireties.Additionally, deprotection conditions can be modified to provide thebest possible yield and purity of siNA constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting XIAP RNA targets. The assaycomprises the system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with XIAP target RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriateXIAP expressing plasmid using T7 RNA polymerase or via chemicalsynthesis as described herein. Sense and antisense siNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 μM final concentration), and 10%[vol/vol]-lysis buffer containing siNA (10 nM final concentration). Thereaction mixture also contains 10 mM creatine phosphate, 10 ug/mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25× Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-⁼P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in theXIAP RNA target for siNA mediated RNAi cleavage, wherein a plurality ofsiNA constructs are screened for RNAi mediated cleavage of the XIAP RNAtarget, for example, by analyzing the assay reaction by electrophoresisof labeled target RNA, or by Northern blotting, as well as by othermethodology well known in the art.

Example 7 Nucleic Acid Inhibition of XIAP Target RNA

siNA molecules targeted to the human XIAP RNA are designed andsynthesized as described above. These nucleic acid molecules can betested for cleavage activity in vivo, for example, using the followingprocedure. The target sequences and the nucleotide location within theXIAP RNA are given in Tables H and III.

Two formats are used to test the efficacy of siNAs targeting XIAP.First, the reagents are tested in cell culture using, for example,cultured human T-cells, A549 cells, or HeLa cells, to determine theextent of RNA and protein inhibition. siNA reagents (e.g.; see Tables IIand III) are selected against the XIAP target as described herein. RNAinhibition is measured after delivery of these reagents by a suitabletransfection agent to, for example, cultured human T-cells, A549 cells,or HeLa cells. Relative amounts of target RNA are measured versus actinusing real-time PCR monitoring of amplification (e.g., ABI 7700TAQMAN®). A comparison is made to a mixture of oligonucleotide sequencesmade to unrelated targets or to a randomized siNA control with the sameoverall length and chemistry, but randomly substituted at each position.Primary and secondary lead reagents are chosen for the target andoptimization performed. After an optimal transfection agentconcentration is chosen, a RNA time-course of inhibition is performedwith the lead siNA molecule. In addition, a cell-plating format can beused to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., cultured human T-cells, A549 cells, or HeLa cells) areseeded, for example, at 1×10⁵ cells per well of a six-well dish in EGM-2(BioWhittaker) the day before transfection. siNA (final concentration,for example 20 nM) and cationic lipid (e.g., final concentration 2μg/ml) are complexed in EGM basal media (Bio Whittaker) at 37° C. for 30minutes in polystyrene tubes. Following vortexing, the complexed siNA isadded to each well and incubated for the times indicated. For initialoptimization experiments, cells are seeded, for example, at 1×10³ in 96well plates and siNA complex added as described. Efficiency of deliveryof siNA to cells is determined using a fluorescent siNA complexed withlipid. Cells in 6-well dishes are incubated with siNA for 24 hours,rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at roomtemperature. Uptake of siNA is visualized using a fluorescentmicroscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer; 900nM reverse primer, 100 nM probe, 1× TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, IOU RNase Inhibitor (Promega), 1.25U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/r×n) and normalizing to β-actin or GAPDH mRNA inparallel TAQMAN® reactions (real-time PCR monitoring of amplification).For each gene of interest an upper and lower primer and a fluorescentlylabeled probe are designed. Real time incorporation of SYBR Green I dyeinto a specific PCR product can be measured in glass capillary tubesusing a lightcyler. A standard curve is generated for each primer pairusing control cRNA. Values are represented as relative expression toGAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8 Animal Models Useful to Evaluate the Down-Regulation of XIAPGene Expression Cell Culture

There are numerous cell culture systems that can be used to analyzereduction of XIAP levels either directly or indirectly by measuringdownstream effects. For example, HELA cells can be used in cell cultureexperiments to assess the efficacy of nucleic acid molecules of theinvention. As such, cells treated with nucleic acid molecules of theinvention (e.g., siNA) targeting XIAP RNA would be expected to havedecreased XIAP expression capacity compared to matched control nucleicacid molecules having a scrambled or inactive sequence. In anon-limiting example, HELA cells are cultured and XIAP expression isquantified, for example by time-resolved immuno fluorometric assay. XIAPmessenger-RNA expression is quantitated with RT-PCR in cultured cells.Untreated cells are compared to cells treated with siNA moleculestransfected with a suitable reagent, for example a cationic lipid suchas lipofectamine, and XIAP protein and RNA levels are quantitated. Doseresponse assays are then performed to establish dose dependentinhibition of XIAP expression. In a non-limiting example, cell cultureexperiments are adapted to those experiments described in Korneluk etal., International PCT Publication No. WO 02/26968.

In several cell culture systems, cationic lipids have been shown toenhance the bioavailability of oligonucleotides to cells in culture(Bennet, et al., 1992, Mol. Pharmacology, 41, 1023-1033). In oneembodiment, siNA molecules of the invention are complexed with cationiclipids for cell culture experiments. siNA and cationic lipid mixturesare prepared in serum-free DMEM immediately prior to addition to thecells. DMEM plus additives are warmed to room temperature (about 20-25°C.) and cationic lipid is added to the final desired concentration andthe solution is vortexed briefly. siNA molecules are added to the finaldesired concentration and the solution is again vortexed briefly andincubated for 10 minutes at room temperature. In dose responseexperiments, the RNA/lipid complex is serially diluted into DMEMfollowing the 10 minute incubation.

Animal Models

Evaluating the efficacy of anti-XIAP agents in animal models is animportant prerequisite to human clinical trials. The role of XIAP hasrecently been investigated (Conte et al., 2001, Proc. Natl. Acad. Sci.USA, 98, 5049) using engineered transgenic mice that over express ahuman XIAP transgene under the control of a T cell specific promoter,lck., to assess the effect of XIAP on T cell development. Theinvestigators evaluated the ability of XIAP to rescueapoptotic-sensitive thymocytes from apoptotic triggers, such as C2ceramide, UV radiation, and anti-Fas antibody. Investigators found thatlck-XIAP thymocytes demonstrated reduced in vitro apoptosis, with only20% cell death relative to untreated lck-xiap thymocytes over 18 hourswhen exposed to C2 ceramide exposure. The ability of XIAP to inhibitapoptotic pathways after exposure to UV radiation and a Fas deathreceptor (anti-Fas antibody) led to the finding that lck-XIAP thymoctyeswere resistant to apoptosis, with apoptosis being reduced compared withwild-type thymocytes (Conte et al., supra).

In addition, thymocytes were treated with dexamethasone or anti-CD3antibody in vitro which triggers apoptosis of thymoctyes; however thelck-XIAP thymocytes demonstrated enhanced resistance to apoptosis.Thymocytes of control mice and lck-xiap mice were also injected withanti-Fas antibody to test levels of apoptosis in vivo. The control micethymocytes had extensive apoptotic death while the thymocytes oflck-XIAP mice had significantly less apoptosis. The resistance toapoptosis by lck-XIAP thymocytes was attributable to over expression ofXIAP (Conte et al., supra).

The animal model described by Conte et al., supra, can be used toevaluate inhibition of XIAP expression and the increased regression oftumor growth after the transfer of conditioned T-cells in the presenceof a XIAP blockade using siNA molecules of the invention. The improvedclearance of tumors in mice can be associated with the XIAP blockadethat improves apoptosis of disease infected cells. These results raisethe possibility that manipulation of XIAP can be used toward therapeuticuse in preventing and/or treating cancer and other proliferativeconditions discussed herein in human subjects.

Example 9 RNAi Mediated Inhibition of XIAP Expression

siNA constructs (Table III) are tested for efficacy in reducing XIAP RNAexpression in, for example, human T-cells, A549 cells, or HeLa cells.Cells are plated approximately 24 hours before transfection in 96-wellplates at 5,000-7,500 cells/well, 100 μl/well, such that at the time oftransfection cells are 70-90% confluent. For transfection, annealedsiNAs are mixed with the transfection reagent (Lipofectamine 2000,Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes atroom temperature. The siNA transfection mixtures are added to cells togive a final siNA concentration of 25 nM in a volume of 150 μl. EachsiNA transfection mixture is added to 3 wells for triplicate siNAtreatments. Cells are incubated at 37° for 24 hours in the continuedpresence of the siNA transfection mixture. At 24 hours, RNA is preparedfrom each well of treated cells. The supernatants with the transfectionmixtures are first removed and discarded, then the cells are lysed andRNA prepared from each well. Target gene expression following treatmentis evaluated by RT-PCR for the target gene and for a control gene (36B4,an RNA polymerase subunit) for normalization. The triplicate data isaveraged and the standard deviations determined for each treatment.Normalized data are graphed and the percent reduction of target mRNA byactive siNAs in comparison to their respective inverted control siNAs isdetermined.

In a non-limiting example, chemically modified siNA constructs (TableIII) were tested for efficacy as described above in XIAP RNA expressionin A549 cells. Active siNAs were evaluated compared to untreated cells,matched chemistry irrelevant controls (IC1, IC2), and a transfectioncontrol. Results are summarized in FIG. 22. FIG. 22 shows results forchemically modified siNA constructs targeting various sites in XIAPmRNA. As shown in FIG. 22, the active siNA constructs providesignificant inhibition of XIAP gene expression in cell cultureexperiments as determined by levels of XIAP mRNA when compared toappropriate controls.

Example 10 Indications

The present body of knowledge in inhibitors of apoptosis researchindicates the need for methods and compounds that can regulate XIAPHIAP1, HIAP2, and/or NAIP gene expression for research, diagnostic, andtherapeutic use. As described herein, the nucleic acid molecules of thepresent invention can be used to treat cancer and other proliferativeconditions such as ovarian cancer; cancers of non-lymphoid parenchymalorgans including the heart, placenta, skeletal muscle and lung; breastcancer, cancers of the head and neck including various lymphomas such asmantle cell lymphoma, non-Hodgkins lymphoma, adenoma, squamous cellcarcinoma, laryngeal carcinoma, cancers of the retina, cancers of theesophagus, multiple myeloma, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, glioblastoma; and proliferativediseases and conditions such as restenosis and polycystic kidneydisease, ocular disease; and any other indications that can respond tothe level of a XIAP, HIAP1, HIAP2, and/or NAIP gene in a cell or tissue.

The use of radiation treatments and chemotherapeutics, such asGemcytabine and cyclophosphamide, are non-limiting examples ofchemotherapeutic agents that can be combined with or used in conjunctionwith the nucleic acid molecules (e.g. siNA molecules) of the instantinvention. Those skilled in the art will recognize that otheranti-cancer compounds and therapies can similarly be readily combinedwith the nucleic acid molecules of the instant invention (e.g. siNAmolecules) and are hence within the scope of the instant invention. Suchcompounds and therapies are well known in the art (see for exampleCancer: Principles and Practice of Oncology, Volumes 1 and 2, edsDevita, V. T., Hellman, S., and Rosenberg, S. A., J. B. LippincottCompany, Philadelphia, USA; incorporated herein by reference) andinclude, without limitation, folates, antifolates, pyrimidine analogs,fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase Iinhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins,platinum analogs, alkylating agents, nitrosoureas, plant derivedcompounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinaseinhibitors, taxols, radiation therapy, surgery, nutritional supplements,gene therapy, radiotherapy, for example 3D-CRT, immunotoxin therapy, forexample ricin, and monoclonal antibodies. Specific examples ofchemotherapeutic compounds that can be combined with or used inconjunction with the nucleic acid molecules of the invention include,but are not limited to, Paclitaxel; Docetaxel; Methotrexate; Doxorubin;Edatrexate; Vinorelbine; Tamoxifen; Leucovorin; 5-fluoro uridine (5-FU);Ionotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin;Mitomycin C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine;L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan;Ifosfamide; 4-hydroperoxycyclophosphamide; Thiotepa; Irinotecan(CAMPTOSAR®, CPT-11, Camptothecin-11, Campto) Tamoxifen; Herceptin; IMCC225; ABX-EGF; and combinations thereof. The above list of compounds arenon-limiting examples of compounds and/or methods that can be combinedwith or used in conjunction with the nucleic acid molecules (e.g. siNA)of the instant invention. Those skilled in the art will recognize thatother drug compounds and therapies can similarly be readily combinedwith the nucleic acid molecules of the instant invention (e.g., siNAmolecules) are hence within the scope of the instant invention.

Example 11 Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with an siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I XIAP Accession Numbers BIRC1 LOCUS NM_004536 6133 bp mRNA linearPRI 05-APR-2003 DEFINITION Homo sapiens baculoviral IAPrepeat-containing 1 (BIRC1), mRNA. ACCESSION NM_004536 BIRC2 LOCUSNM_001166 3496 bp mRNA linear PRI 03-APR-2003 DEFINITION Homo sapiensbaculoviral IAP repeat-containing 2 (BIRC2), mRNA. ACCESSION NM_001166BIRC3 LOCUS NM_001165 3165 bp mRNA linear PRI 03-APR-2003 DEFINITIONHomo sapiens baculoviral IAP repeat-containing 3 (BIRC3), mRNA.ACCESSION NM_001165 BIRC4 LOCUS NM_001167 8413 bp mRNA linear PRI11-JUL-2003 DEFINITION Homo sapiens baculoviral IAP repeat-containing 4(BIRC4), mRNA. ACCESSION NM_001167 BIRC5 LOCUS NM_001168 1619 bp mRNAlinear PRI 03-APR-2003 DEFINITION Homo sapiens baculoviral IAPrepeat-containing 5 (survivin) (BIRC5), mRNA. ACCESSION NM_001168 BIRC6LOCUS NM_016252 14490 bp  mRNA linear PRI 06-APR-2003 DEFINITION Homosapiens baculoviral IAP repeat-containing 6 (apollon) (BIRC6), mRNA.ACCESSION NM_016252 BIRC7-1 LOCUS NM_139317 1322 bp mRNA linear PRI06-APR-2003 DEFINITION Homo sapiens baculoviral IAP repeat-containing 7(livin) (BIRC7), transcript variant 1, mRNA. ACCESSION NM_139317 BIRC7-2LOCUS NM_022161 1268 bp mRNA linear PRI 06-APR-2003 DEFINITION Homosapiens baculoviral IAP repeat-containing 7 (livin) (BIRC7), transcriptvariant 2, mRNA. ACCESSION NM_022161 BIRC8 LOCUS NM_033341 2032 bp mRNAlinear PRI 06-APR-2003 DEFINITION Homo sapiens baculoviral IAPrepeat-containing 8 (BIRC8), mRNA. ACCESSION NM_033341 VERSIONNM_033341.2 GI: 16974127

TABLE II XIAP/BIRC4 siNA AND TARGET SEQUENCES XIAP BIRC4|NM_001167.2 SeqSeq Seq Pos Seq ID UPos Upper seq ID LPos Lower seq ID    3UCCAGAUUGGGGCUCGGGC 1 3 UCCAGAUUGGGGCUCGGGC 1 21 GCCCGAGCCCCAAUCUGGA 468  21 CCGCGCCUCCUCCGGGACC 2 21 CCGCGCCUCCUCCGGGACC 2 39GGUCCCGGAGGAGGCGCGG 469   39 CCUCCCCUUGGACCGAGCC 3 39CCUCCCCUUGGACCGAGCC 3 57 GGCUCGGUCCAAGGGGAGG 470   57CGAUCGCCGCGGGGCAGUU 4 57 CGAUCGCCGCGGGGCAGUU 4 75 AACUGCCCCGCGGCGAUCG471   75 UCGGGCCGGCUGUCCUGGC 5 75 UCGGGCCGGCUGUCCUGGC 5 93GCCAGGACAGCCGGCCCGA 472   93 CGCGAAAAGGUGGACAAGU 6 93CGCGAAAAGGUGGACAAGU 6 111 ACUUGUCCACCUUUUCGCG 473  111UCCUAUUUUCAAGAGAAGA 7 111 UCCUAUUUUCAAGAGAAGA 7 129 UCUUCUCUUGAAAAUAGGA474  129 AUGACUUUUAACAGUUUUG 8 129 AUGACUUUUAACAGUUUUG 8 147CAAAACUGUUAAAAGUCAU 475  147 GAAGGAUCUAAAACUUGUG 9 147GAAGGAUCUAAAACUUGUG 9 165 CACAAGUUUUAGAUCCUUC 476  165GUACCUGCAGACAUCAAUA 10 165 GUACCUGCAGACAUCAAUA 10 183UAUUGAUGUGUGCAGGUAC 477  183 AAGGAAGAAGAAUUUGUAG 11 183AAGGAAGAAGAAUUUGUAG 11 201 CUACAAAUUCUUCUUCCUU 478  201GAAGAGUUUAAUAGAUUAA 12 201 GAAGAGUUUAAUAGAUUAA 12 219UUAAUCUAUUAAACUCUUC 479  219 AAAACUUUUGCUAAUUUUC 13 219AAAACUUUUGCUAAUUUUC 13 237 GAAAAUUAGCAAAAGUUUU 480  237CCAAGUGGUAGUCCUGUUU 14 237 CCAAGUGGUAGUCCUGUUU 14 255AAACAGGACUACCACUUGG 481  255 UCAGCAUCAACACUGGCAC 15 255UCAGCAUCAACACUGGCAC 15 273 GUGCCAGUGUUGAUGCUGA 482  273CGAGCAGGGUUUCUUUAUA 16 273 CGAGCAGGGUUUCUUUAUA 16 291UAUAAAGAAACCCUGCUCG 483  291 ACUGGUGAAGGAGAUACCG 17 291ACUGGUGAAGGAGAUACCG 17 309 CGGUAUCUCCUUCACCAGU 484  309GUGCGGUGCUUUAGUUGUC 18 309 GUGCGGUGCUUUAGUUGUC 18 327GACAACUAAAGCACCGCAC 485  327 CAUGCAGCUGUAGAUAGAU 19 327CAUGCAGCUGUAGAUAGAU 19 345 AUCUAUCUACAGCUGCAUG 486  345UGGCAAUAUGGAGACUCAG 20 345 UGGCAAUAUGGAGACUCAG 20 363CUGAGUCUCCAUAUUGCCA 487  363 GCAGUUGGAAGACACAGGA 21 363GCAGUUGGAAGACACAGGA 21 381 UCCUGUGUCUUCCAACUGC 488  381AAAGUAUCCCCAAAUUGCA 22 381 AAAGUAUCCCCAAAUUGCA 22 399UGCAAUUUGGGGAUACUUU 489  399 AGAUUUAUCAACGGCUUUU 23 399AGAUUUAUCAACGGCUUUU 23 417 AAAAGCCGUUGAUAAAUCU 490  417UAUCUUGAAAAUAGUGCCA 24 417 UAUCUUGAAAAUAGUGCCA 24 435UGGCACUAUUUUCAAGAUA 491  435 ACGCAGUCUACAAAUUCUG 25 435ACGCAGUCUACAAAUUCUG 25 453 CAGAAUUUGUAGACUGCGU 492  453GGUAUCCAGAAUGGUCAGU 26 453 GGUAUCCAGAAUGGUCAGU 26 471ACUGACCAUUCUGGAUACC 493  471 UACAAAGUUGAAAACUAUC 27 471UACAAAGUUGAAAACUAUC 27 489 GAUAGUUUUCAACUUUGUA 494  489CUGGGAAGCAGAGAUCAUU 28 489 CUGGGAAGCAGAGAUCAUU 28 507AAUGAUCUCUGCUUCCCAG 495  507 UUUGCCUUAGACAGGCCAU 29 507UUUGCCUUAGACAGGCCAU 29 525 AUGGCCUGUCUAAGGCAAA 496  525UCUGAGACACAUGCAGACU 30 525 UCUGAGACACAUGCAGACU 30 543AGUCUGCAUGUGUCUCAGA 497  543 UAUCUUUUGAGAACUGGGC 31 543UAUCUUUUGAGAACUGGGC 31 561 GCCCAGUUCUCAAAAGAUA 498  561CAGGUUGUAGAUAUAUCAG 32 561 CAGGUUGUAGAUAUAUCAG 32 579CUGAUAUAUCUACAACCUG 499  579 GACACCAUAUACCCGAGGA 33 579GACACCAUAUACCCGAGGA 33 597 UCCUCGGGUAUAUGGUGUC 500  597AACCCUGCCAUGUAUAGUG 34 597 AACCCUGCCAUGUAUAGUG 34 615CACUAUACAUGGCAGGGUU 501  615 GAAGAAGCUAGAUUAAAGU 35 615GAAGAAGCUAGAUUAAAGU 35 633 ACUUUAAUCUAGCUUCUUC 502  633UCCUUUCAGAACUGGCCAG 36 633 UCCUUUCAGAACUGGCCAG 36 651CUGGCCAGUUCUGAAAGGA 503  651 GACUAUGCUCACCUAACCC 37 651GACUAUGCUCACCUAACCC 37 669 GGGUUAGGUGAGCAUAGUC 504  669CCAAGAGAGUUAGCAAGUG 38 669 CCAAGAGAGUUAGCAAGUG 38 687CACUUGCUAACUCUCUUGG 505  687 GCUGGACUCUACUACACAG 39 687GCUGGACUCUACUACACAG 39 705 CUGUGUAGUAGAGUCCAGC 506  705GGUAUUGGUGACCAAGUGC 40 705 GGUAUUGGUGACCAAGUGC 40 723GCACUUGGUCACCAAUACC 507  723 CAGUGCUUUUGUUGUGGUG 41 723CAGUGCUUUUGUUGUGGUG 41 741 CACCACAACAAAAGCACUG 508  741GGAAAACUGAAAAAUUGGG 42 741 GGAAAACUGAAAAAUUGGG 42 759CCCAAUUUUUCAGUUUUCC 509  759 GAACCUUGUGAUCGUGCCU 43 759GAACCUUGUGAUCGUGCCU 43 777 AGGCACGAUCACAAGGUUC 510  777UGGUCAGAACACAGGCGAC 44 777 UGGUCAGAACACAGGCGAC 44 795GUCGCCUGUGUUCUGACCA 511  795 CACUUUCCUAAUUGCUUCU 45 795CACUUUCCUAAUUGCUUCU 45 813 AGAAGCAAUUAGGAAAGUG 512  813UUUGUUUUGGGCCGGAAUC 46 813 UUUGUUUUGGGCCGGAAUC 46 831GAUUCCGGCCCAAAACAAA 513  831 CUUAAUAUUCGAAGUGAAU 47 831CUUAAUAUUCGAAGUGAAU 47 849 AUUCACUUCGAAUAUUAAG 514  849UCUGAUGCUGUGAGUUCUG 48 849 UCUGAUGCUGUGAGUUCUG 48 867CAGAACUCACAGCAUCAGA 515  867 GAUAGGAAUUUCCCAAAUU 49 867GAUAGGAAUUUCCCAAAUU 49 885 AAUUUGGGAAAUUCCUAUC 516  885UCAACAAAUCUUCCAAGAA 50 885 UCAACAAAUCUUCCAAGAA 50 903UUCUUGGAAGAUUUGUUGA 517  903 AAUCCAUCCAUGGCAGAUU 51 903AAUCCAUCCAUGGCAGAUU 51 921 AAUCUGCCAUGGAUGGAUU 518  921UAUGAAGCACGGAUCUUUA 52 921 UAUGAAGCACGGAUCUUUA 52 939UAAAGAUCCGUGCUUCAUA 519  939 ACUUUUGGGACAUGGAUAU 53 939ACUUUUGGGACAUGGAUAU 53 957 AUAUCCAUGUCCCAAAAGU 520  957UACUCAGUUAACAAGGAGC 54 957 UACUCAGUUAACAAGGAGC 54 975GCUCCUUGUUAACUGAGUA 521  975 CAGCUUGCAAGAGCUGGAU 55 975CAGCUUGCAAGAGCUGGAU 55 993 AUCCAGCUCUUGCAAGCUG 522  993UUUUAUGCUUUAGGUGAAG 56 993 UUUUAUGCUUUAGGUGAAG 56 1011CUUCACCUAAAGCAUAAAA 523 1011 GGUGAUAAAGUAAAGUGCU 57 1011GGUGAUAAAGUAAAGUGCU 57 1029 AGCACUUUACUUUAUCACC 524 1029UUUCACUGUGGAGGAGGGC 58 1029 UUUCACUGUGGAGGAGGGC 58 1047GCCCUCCUCCACAGUGAAA 525 1047 CUAACUGAUUGGAAGCCCA 59 1047CUAACUGAUUGGAAGCCCA 59 1065 UGGGCUUCCAAUCAGUUAG 526 1065AGUGAAGACCCUUGGGAAC 60 1065 AGUGAAGACCCUUGGGAAC 60 1083GUUCCCAAGGGUCUUCACU 527 1083 CAACAUGCUAAAUGGUAUC 61 1083CAACAUGCUAAAUGGUAUC 61 1101 GAUACCAUUUAGCAUGUUG 528 1101CCAGGGUGCAAAUAUCUGU 62 1101 CCAGGGUGCAAAUAUCUGU 62 1119ACAGAUAUUUGCACCCUGG 529 1119 UUAGAACAGAAGGGACAAG 63 1119UUAGAACAGAAGGGACAAG 63 1137 CUUGUCCCUUCUGUUCUAA 530 1137GAAUAUAUAAACAAUAUUC 64 1137 GAAUAUAUAAACAAUAUUC 64 1155GAAUAUUGUUUAUAUAUUC 531 1155 CAUUUAACUCAUUCACUUG 65 1155CAUUUAACUCAUUCACUUG 65 1173 CAAGUGAAUGAGUUAAAUG 532 1173GAGGAGUGUCUGGUAAGAA 66 1173 GAGGAGUGUCUGGUAAGAA 66 1191UUCUUACCAGACACUCCUC 533 1191 ACUACUGAGAAAACACCAU 67 1191ACUACUGAGAAAACACCAU 67 1209 AUGGUGUUUUCUCAGUAGU 534 1209UCACUAACUAGAAGAAUUG 68 1209 UCACUAACUAGAAGAAUUG 68 1227CAAUUCUUCUAGUUAGUGA 535 1227 GAUGAUACCAUCUUCCAAA 69 1227GAUGAUACCAUCUUCCAAA 69 1245 UUUGGAAGAUGGUAUCAUC 536 1245AAUCCUAUGGUACAAGAAG 70 1245 AAUCCUAUGGUACAAGAAG 70 1263CUUCUUGUACCAUAGGAUU 537 1263 GCUAUACGAAUGGGGUUCA 71 1263GCUAUACGAAUGGGGUUCA 71 1281 UGAACCCCAUUCGUAUAGC 538 1281AGUUUCAAGGACAUUAAGA 72 1281 AGUUUCAAGGACAUUAAGA 72 1299UCUUAAUGUCCUUGAAACU 539 1299 AAAAUAAUGGAGGAAAAAA 73 1299AAAAUAAUGGAGGAAAAAA 73 1317 UUUUUUCCUCCAUUAUUUU 540 1317AUUCAGAUAUGUGGGAGCA 74 1317 AUUCAGAUAUCUGGGAGCA 74 1335UGCUCCCAGAUAUCUGAAU 541 1335 AACUAUAAAUCACUUGAGG 75 1335AACUAUAAAUCACUUGAGG 75 1353 CCUCAAGUGAUUUAUAGUU 542 1353GUUCUGGUUGCAGAUCUAG 76 1353 GUUCUGGUUGCAGAUCUAG 76 1371CUAGAUCUGCAACCAGAAC 543 1371 GUGAAUGCUCAGAAAGACA 77 1371GUGAAUGCUCAGAAAGACA 77 1389 UGUCUUUCUGAGCAUUCAC 544 1389AGUAUGCAAGAUGAGUCAA 78 1389 AGUAUGCAAGAUGAGUCAA 78 1407UUGACUCAUCUUGCAUACU 545 1407 AGUCAGACUUCAUUACAGA 79 1407AGUCAGACUUCAUUACAGA 79 1425 UCUGUAAUGAAGUCUGACU 546 1425AAAGAGAUUAGUACUGAAG 80 1425 AAAGAGAUUAGUACUGAAG 80 1443CUUCAGUACUAAUCUCUUU 547 1443 GAGCAGCUAAGGCGCCUGC 81 1443GAGCAGCUAAGGCGCCUGC 81 1461 GCAGGCGCCUUAGCUGCUC 548 1461CAAGAGGAGAAGCUUUGCA 82 1461 CAAGAGGAGAAGCUUUGCA 82 1479UGCAAAGCUUCUCCUCUUG 549 1479 AAAAUCUGUAUGGAUAGAA 83 1479AAAAUCUGUAUGGAUAGAA 83 1497 UUCUAUCCAUACAGAUUUU 550 1497AAUAUUGCUAUCGUUUUUG 84 1497 AAUAUUGCUAUCGUUUUUG 84 1515CAAAAACGAUAGCAAUAUU 551 1515 GUUCCUUGUGGACAUCUAG 85 1515GUUCCUUGUGGACAUCUAG 85 1533 CUAGAUGUCCACAAGGAAC 552 1533GUCACUUGUAAACAAUGUG 86 1533 GUCACUUGUAAACAAUGUG 86 1551CACAUUGUUUACAAGUGAC 553 1551 GCUGAAGCAGUUGACAAGU 87 1551GCUGAAGCAGUUGACAAGU 87 1569 ACUUGUCAACUGCUUCAGC 554 1569UGUCCCAUGUGCUACACAG 88 1569 UGUCCCAUGUGCUACACAG 88 1587CUGUGUAGCACAUGGGACA 555 1587 GUCAUUACUUUCAAGCAAA 89 1587GUCAUUACUUUCAAGCAAA 89 1605 UUUGCUUGAAAGUAAUGAC 556 1605AAAAUUUUUAUGUCUUAAU 90 1605 AAAAUUUUUAUGUCUUAAU 90 1623AUUAAGACAUAAAAAUUUU 557 1623 UGUAACUCUAUAGUAGGCA 91 1623UCUAACUCUAUAGUAGGCA 91 1641 UGCCUACUAUAGAGUUAGA 558 1641AUGUUAUGUUGUUCUUAUU 92 1641 AUGUUAUGUUGUUCUUAUU 92 1659AAUAAGAACAACAUAACAU 559 1659 UACCCUGAUUGAAUGUGUG 93 1659UACCCUGAUUGAAUGUGUG 93 1677 CACACAUUCAAUCAGGGUA 560 1677GAUGUGAACUGACUUUAAG 94 1677 GAUGUGAACUGACUUUAAG 94 1695CUUAAAGUCAGUUCACAUC 561 1695 GUAAUCAGGAUUGAAUUCC 95 1695GUAAUCAGGAUUGAAUUCC 95 1713 GGAAUUCAAUCCUGAUUAC 562 1713CAUUAGCAUUUGCUACCAA 96 1713 CAUUAGCAUUUGCUACCAA 96 1731UUGGUAGCAAAUGCUAAUG 563 1731 AGUAGGAAAAAAAAUGUAC 97 1731AGUAGGAAAAAAAAUGUAC 97 1749 GUACAUUUUUUUUCCUACU 564 1749CAUGGCAGUGUUUUAGUUG 98 1749 CAUGGCAGUGUUUUAGUUG 98 1767CAACUAAAACACUGCCAUG 565 1767 GGCAAUAUAAUCUUUGAAU 99 1767GGCAAUAUAAUCUUUGAAU 99 1785 AUUCAAAGAUUAUAUUGCC 566 1785UUUCUUGAUUUUUCAGGGU 100 1785 UUUCUUGAUUUUUCAGGGU 100 1803ACCCUGAAAAAUCAAGAAA 567 1803 UAUUAGCUGUAUUAUCCAU 101 1803UAUUAGCUGUAUUAUCCAU 101 1821 AUGGAUAAUACAGCUAAUA 568 1821UUUUUUUUACUGUUAUUUA 102 1821 UUUUUUUUACUGUUAUUUA 102 1839UAAAUAACAGUAAAAAAAA 569 1839 AAUUGAAACCAUAGACUAA 103 1839AAUUGAAACCAUAGACUAA 103 1857 UUAGUCUAUGGUUUCAAUU 570 1857AGAAUAAGAAGCAUCAUAC 104 1857 AGAAUAAGAAGCAUCAUAC 104 1875GUAUGAUGCUUCUUAUUCU 571 1875 CUAUAACUGAACACAAUGU 105 1875CUAUAACUGAACACAAUGU 105 1893 ACAUUGUGUUCAGUUAUAG 572 1893UGUAUUCAUAGUAUACUGA 106 1893 UGUAUUCAUAGUAUACUGA 106 1911UCAGUAUACUAUGAAUACA 573 1911 AUUUAAUUUCUAAGUGUAA 107 1911AUUUAAUUUCUAAGUGUAA 107 1929 UUACACUUAGAAAUUAAAU 574 1929AGUGAAUUAAUCAUCUGGA 108 1929 AGUGAAUUAAUCAUCUGGA 108 1947UCCAGAUGAUUAAUUCACU 575 1947 AUUUUUUAUUCUUUUCAGA 109 1947AUUUUUUAUUCUUUUCAGA 109 1965 UCUGAAAAGAAUAAAAAAU 576 1965AUAGGCUUAACAAAUGGAG 110 1965 AUAGGCUUAACAAAUGGAG 110 1983CUCCAUUUGUUAAGCCUAU 577 1983 GCUUUCUGUAUAUAAAUGU 111 1983GCUUUCUGUAUAUAAAUGU 111 2001 ACAUUUAUAUACAGAAAGC 578 2001UGGAGAUUAGAGUUAAUCU 112 2001 UGGAGAUUAGAGUUAAUCU 112 2019AGAUUAACUCUAAUCUCCA 579 2019 UCCCCAAUCACAUAAUUUG 113 2019UCCCCAAUCACAUAAUUUG 113 2037 CAAAUUAUGUGAUUGGGGA 580 2037GUUUUGUGUGAAAAAGGAA 114 2037 GUUUUGUGUGAAAAAGGAA 114 2055UUCCUUUUUCACACAAAAC 581 2055 AUAAAUUGUUCCAUGCUGG 115 2055AUAAAUUGUUCCAUGCUGG 115 2073 CCAGCAUGGAACAAUUUAU 582 2073GUGGAAAGAUAGAGAUUGU 116 2073 GUGGAAAGAUAGAGAUUGU 116 2091ACAAUCUCUAUCUUUCCAC 583 2091 UUUUUAGAGGUUGGUUGUU 117 2091UUUUUAGAGGUUGGUUGUU 117 2109 AACAACCAACCUCUAAAAA 584 2109UGUGUUUUAGGAUUCUGUC 118 2109 UGUGUUUUAGGAUUCUGUC 118 2127GACAGAAUCCUAAAACACA 585 2127 CCAUUUUCUUUUAAAGUUA 119 2127CCAUUUUCUUUUAAAGUUA 119 2145 UAACUUUAAAAGAAAAUGG 586 2145AUAAACACGUAGUUGUGCG 120 2145 AUAAACACGUAGUUGUGCG 120 2163CGCACAAGUACGUGUUUAU 587 2163 GAAUUAUUUUUUUAAAGUG 121 2163GAAUUAUUUUUUUAAAGUG 121 2181 CACUUUAAAAAAAUAAUUC 588 2181GAUUUGCCAUUUUUGAAAG 122 2181 GAUUUGCCAUUUUUGAAAG 122 2199CUUUCAAAAAUGGCAAAUC 589 2199 GCGUAUUUAAUGAUAGAAU 123 2199GCGUAUUUAAUGAUAGAAU 123 2217 AUUCUAUCAUUAAAUACGC 590 2217UACUAUCGAGCCAACAUGU 124 2217 UACUAUCGAGCCAACAUGU 124 2235ACAUGUUGGCUCGAUAGUA 591 2235 UACUGACAUGGAAAGAUGU 125 2235UACUGACAUGGAAAGAUGU 125 2253 ACAUCUUUCCAUGUCAGUA 592 2253UCAAAGAUAUGUUAAGUGU 126 2253 UCAAAGAUAUGUUAAGUGU 126 2271ACACUUAACAUAUCUUUGA 593 2271 UAAAAUGCAAGUGGCAAAA 127 2271UAAAAUGCAAGUGGCAAAA 127 2289 UUUUGCCACUUGCAUUUUA 594 2289ACACUAUGUAUAGUCUGAG 128 2289 ACACUAUGUAUAGUCUGAG 128 2307CUCAGACUAUACAUAGUGU 595 2307 GCCAGAUCAAAGUAUGUAU 129 2307GCCAGAUCAAAGUAUGUAU 129 2325 AUACAUACUUUGAUCUGGC 596 2325UGUUUUUAAUAUGCAUAGA 130 2325 UGUUUUUAAUAUGCAUAGA 130 2343UCUAUGCAUAUUAAAAACA 597 2343 AACAAAAGAUUUGGAAAGA 131 2343AACAAAAGAUUUGGAAAGA 131 2361 UCUUUCCAAAUCUUUUGUU 598 2361AUAUACACCAAACUGUUAA 132 2361 AUAUACACCAAACUGUUAA 132 2379UUAACAGUUUGGUGUAUAU 599 2379 AAUGUGGUUUCUGUUCGGG 133 2379AAUGUGGUUUCUGUUCGGG 133 2397 CCCGAAGAGAAACCACAUU 600 2397GGAGGGGGGGAUUGGGGGA 134 2397 GGAGGGGGGGAUUGGGGGA 134 2415UCCCCCAAUCCCCCCCUCC 601 2415 AGGGGCCCCAGAGGGGUUU 135 2415AGGGGCCCCAGAGGGGUUU 135 2433 AAACCCCUCUGGGGCCCCU 602 2433UUAUAGGGGCCUUUUCACU 136 2433 UUAUAGGGGCCUUUUCACU 136 2451AGUGAAAAGGCCCCUAUAA 603 2451 UUUCUACUUUUUUCAUUUU 137 2451UUUCUACUUUUUUCAUUUU 137 2469 AAAAUGAAAAAAGUAGAAA 604 2469UGUUCUGUUCGAAUUUUUU 138 2469 UGUUCUGUUCGAAUUUUUU 138 2487AAAAAAUUCGAACAGAACA 605 2487 UAUAAGUAUGUAUUACUUU 139 2487UAUAAGUAUGUAUUACUUU 139 2505 AAAGUAAUACAUACUUAUA 606 2505UUGUAAUCAGAAUUUUUAG 140 2505 UUGUAAUCAGAAUUUUUAG 140 2523CUAAAAAUUCUGAUUACAA 607 2523 GAAAGUAUUUUGCUGAUUU 141 2523GAAAGUAUUUUGCUGAUUU 141 2541 AAAUCAGCAAAAUACUUUC 608 2541UAAAGGCUUAGGCAUGUUC 142 2541 UAAAGGCUUAGGCAUGUUC 142 2559GAACAUGCCUAAGCCUUUA 609 2559 CAAACGCCUGCAAAACUAC 143 2559CAAACGCCUGCAAAACUAC 143 2577 GUAGUUUUGCAGGCGUUUG 610 2577CUUAUCACUCAGCUUUAGU 144 2577 CUUAUCACUCAGCUUUAGU 144 2595ACUAAAGCUGAGUGAUAAG 611 2595 UUUUUCUAAUCCAAGAAGG 145 2595UUUUUCUAAUCCAAGAAGG 145 2613 CCUUCUUGGAUUAGAAAAA 612 2613GCAGGGCAGUUAACCUUUU 146 2613 GCAGGGCAGUUAACCUUUU 146 2631AAAAGGUUAACUGCCCUGC 613 2631 UUGGUGCCAAUGUGAAAUG 147 2631UUGGUGCCAAUGUGAAAUG 147 2649 CAUUUCACAUUGGCACCAA 614 2649GUAAAUGAUUUUAUGUUUU 148 2649 GUAAAUGAUUUUAUGUUUU 148 2667AAAACAUAAAAUCAUUUAC 615 2667 UUCCUGCUUUGUGGAUGAA 149 2667UUCCUGCUUUGUGGAUGAA 149 2685 UUCAUCCACAAAGCAGGAA 616 2685AAAAUAUUUCUGAGUGGUA 150 2685 AAAAUAUUUCUGAGUGGUA 150 2703UACCACUCAGAAAUAUUUU 617 2703 AGUUUUUUGACAGGUAGAC 151 2703AGUUUUUUGACAGGUAGAC 151 2721 GUCUACCUGUCAAAAAACU 618 2721CCAUGUCUUAUCUUGUUUC 152 2721 CCAUGUCUUAUCUUGUUUC 152 2739GAAACAAGAUAAGACAUGG 619 2739 CAAAAUAAGUAUUUCUGAU 153 2739CAAAAUAAGUAUUUCUGAU 153 2757 AUCAGAAAUACUUAUUUUG 620 2757UUUUGUAAAAUGAAAUAUA 154 2757 UUUUGUAAAAUGAAAUAUA 154 2775UAUAUUUCAUUUUACAAAA 621 2775 AAAAUAUGUCUCAGAUCUU 155 2775AAAAUAUGUCUCAGAUCUU 155 2793 AAGAUCUGAGACAUAUUUU 622 2793UCCAAUUAAUUAGUAAGGA 156 2793 UCCAAUUAAUUAGUAAGGA 156 2811UCCUUACUAAUUAAUUGGA 623 2811 AUUCAUCCUUAAUCCUUGC 157 2811AUUCAUCCUUAAUCCUUGC 157 2829 GCAAGGAUUAAGGAUGAAU 624 2829CUAGUUUAAGCCUGCCUAA 158 2829 CUAGUUUAAGCCUGCCUAA 158 2847UUAGGCAGGCUUAAACUAG 625 2847 AGUCACUUUACUAAAAGAU 159 2847AGUCACUUUACUAAAAGAU 159 2865 AUCUUUUAGUAAAGUGACU 626 2865UCUUUGUUAACUCAGUAUU 160 2865 UCUUUGUUAACUCAGUAUU 160 2883AAUACUGAGUUAACAAAGA 627 2883 UUUAAACAUCUGUCAGCUU 161 2883UUUAAACAUCUGUCAGCUU 161 2901 AAGCUGACAGAUGUUUAAA 628 2901UAUGUAGGUAAAAGUAGAA 162 2901 UAUGUAGGUAAAAGUAGAA 162 2919UUCUACUUUUACCUACAUA 629 2919 AGCAUGUUUGUACACUGCU 163 2919AGCAUGUUUGUACACUGCU 163 2937 AGCAGUGUACAAACAUGCU 630 2937UUGUAGUUAUAGUGACAGC 164 2937 UUGUAGUUAUAGUGACAGC 164 2955GCUGUCACUAUAACUACAA 631 2955 CUUUCCAUGUUGAGAUUCU 165 2955CUUUCCAUGUUGAGAUUCU 165 2973 AGAAUCUCAACAUGGAAAG 632 2973UCAUAUCAUCUUGUAUCUU 166 2973 UCAUAUCAUCUUGUAUCUU 166 2991AAGAUACAAGAUGAUAUGA 633 2991 UAAAGUUUCAUGUGAGUUU 167 2991UAAAGUUUCAUGUGAGUUU 167 3009 AAACUCACAUGAAACUUUA 634 3009UUUACCGUUAGGAUGAUUA 168 3009 UUUACCGUUAGGAUGAUUA 168 3027UAAUCAUCCUAACGGUAAA 635 3027 AAGAUGUAUAUAGGACAAA 169 3027AAGAUGUAUAUAGGACAAA 169 3045 UUUGUCCUAUAUACAUCUU 636 3045AAUGUUAAGUCUUUCCUCU 170 3045 AAUGUUAAGUCUUUCCUCU 170 3063AGAGGAAAGACUUAACAUU 637 3063 UACCUACAUUUGUUUUCUU 171 3063UACCUACAUUUGUUUUCUU 171 3081 AAGAAAACAAAUGUAGGUA 638 3081UGGCUAGUAAUAGUAGUAG 172 3081 UGGCUAGUAAUAGUAGUAG 172 3099CUACUACUAUUACUAGCCA 639 3099 GAUACUUCUGAAAUAAAUG 173 3099GAUACUUCUGAAAUAAAUG 173 3117 CAUUUAUUUCAGAAGUAUC 640 3117GUUCUCUCAAGAUCCUUAA 174 3117 GUUCUCUCAAGAUCCUUAA 174 3135UUAAGGAUCUUGAGAGAAC 641 3135 AAACCUCUUGGAAAUUAUA 175 3135AAACCUCUUGGAAAUUAUA 175 3153 UAUAAUUUCCAAGAGGUUU 642 3153AAAAAUAUUGGCAAGAAAA 176 3153 AAAAAUAUUGGCAAGAAAA 176 3171UUUUCUUGCCAAUAUUUUU 643 3171 AGAAGAAUAGUUGUUUAAA 177 3171AGAAGAAUAGUUGUUUAAA 177 3189 UUUAAACAACUAUUCUUCU 644 3189AUAUUUUUUAAAAAACACU 178 3189 AUAUUUUUUAAAAAACACU 178 3207AGUGUUUUUUAAAAAAUAU 645 3207 UUGAAUAAGAAUCAGUAGG 179 3207UUGAAUAAGAAUCAGUAGG 179 3225 CCUACUGAUUCUUAUUCAA 646 3225GGUAUAAACUAGAAGUUUA 180 3225 GGUAUAAACUAGAAGUUUA 180 3243UAAACUUCUAGUUUAUACC 647 3243 AAAAAUGCUUCAUAGAACG 181 3243AAAAAUGCUUCAUAGAACG 181 3261 CGUUCUAUGAAGCAUUUUU 648 3261GUCCAGGGUUUACAUUACA 182 3261 GUCCAGGGUUUACAUUACA 182 3279UGUAAUGUAAACCCUGGAC 649 3279 AAGAUUCUCACAACAAACC 183 3279AAGAUUCUCACAACAAACC 183 3297 GGUUUGUUGUGAGAAUCUU 650 3297CUAUUGUAGAGGUGAGUAA 184 3297 CUAUUGUAGAGGUGAGUAA 184 3315UUACUCACCUCUACAAUAG 651 3315 AGGCAUGUUACUACAGAGG 185 3315AGGCAUGUUACUACAGAGG 185 3333 CCUCUGUAGUAACAUGCCU 652 3333GAAAGUUUGAGAGUAAAAC 186 3333 GAAAGUUUGAGAGUAAAAC 186 3351GUUUUACUCUCAAACUUUC 653 3351 CUGUAAAAAAUUAUAUUUU 187 3351CUGUAAAAAAUUAUAUUUU 187 3369 AAAAUAUAAUUUUUUACAG 654 3369UUGUUGUACUUUCUAAGAG 188 3369 UUGUUGUACUUUCUAAGAG 188 3387CUCUUAGAAAGUACAACAA 655 3387 GAAAGAGUAUUGUUAUGUU 189 3387GAAAGAGUAUUGUUAUGUU 189 3405 AACAUAACAAUACUCUUUC 656 3405UCUCCUAACUUCUGUUGAU 190 3405 UCUCCUAACUUCUGUUGAU 190 3423AUCAACAGAAGUUAGGAGA 657 3423 UUACUACUUUAAGUGAUAU 191 3423UUACUACUUUAAGUGAUAU 191 3441 AUAUCACUUAAAGUAGUAA 658 3441UUCAUUUAAAACAUUGCAA 192 3441 UUCAUUUAAAACAUUGCAA 192 3459UUGCAAUGUUUUAAAUGAA 659 3459 AAUUUAUUUUAUUUAUUUA 193 3459AAUUUAUUUUAUUUAUUUA 193 3477 UAAAUAAAUAAAAUAAAUU 660 3477AAUUUUCUUUUUGAGAUGG 194 3477 AAUUUUCUUUUUGAGAUGG 194 3495CCAUCUCAAAAAGAAAAUU 661 3495 GAGUCUUGCUUGUCACCCA 195 3495GAGUCUUGCUUGUCACCCA 195 3513 UGGGUGACAAGCAAGACUC 662 3513AGGCUGGAGUGCAGUGGAG 196 3513 AGGCUGGAGUGCAGUGGAG 196 3531CUCCACUGCACUCCAGCCU 663 3531 GUGAUCUCUGCUCACUGCA 197 3531GUGAUCUCUGCUCACUGCA 197 3549 UGCAGUGAGCAGAGAUCAC 664 3549AACCUCCGCCUUCUGGGUU 198 3549 AACCUCCGCCUUCUGGGUU 198 3567AACCCAGAAGGCGGAGGUU 665 3567 UCAAGCGAUUCUCGUGCCU 199 3567UCAAGCGAUUCUCGUGCCU 199 3585 AGGCACGAGAAUCGCUUGA 666 3585UCAGCUUCCUGAGUAGCUG 200 3585 UCAGCUUCCUGAGUAGCUG 200 3603CAGCUACUCAGGAAGCUGA 667 3603 GGAAUUACAGGCAGGUGCC 201 3603GGAAUUACAGGCAGGUGCC 201 3621 GGCACCUGCCUGUAAUUCC 668 3621CACCAUGCCCGACUAAUUU 202 3621 CACCAUGCCCGACUAAUUU 202 3639AAAUUAGUCGGGCAUGGUG 669 3639 UUUUUUUAUUUUUAGUAGA 203 3639UUUUUUUAUUUUUAGUAGA 203 3657 UCUACUAAAAAUAAAAAAA 670 3657AGACGGGGUUUCACCAUGU 204 3657 AGACGGGGUUUCACCAUGU 204 3675ACAUGGUGAAACCCCGUCU 671 3675 UUGGCCAGGCUGGUAUCAA 205 3675UUGGCCAGGCUGGUAUCAA 205 3693 UUGAUACCAGCCUGGCCAA 672 3693AACUCCUGACCUCAAGAGA 206 3693 AACUCCUGACCUCAAGAGA 206 3711UCUCUUGAGGUCAGGAGUU 673 3711 AUCCACUCGCCUUGCCCUC 207 3711AUCCACUCGCCUUGCCCUC 207 3729 GAGGGCAAGGCGAGUGGAU 674 3729CCCAAAGUGCUGGGAUUAC 208 3729 CCCAAAGUGCUGGGAUUAC 208 3747GUAAUCCCAGCACUUUGGG 675 3747 CAGGCUUGAGCCACCACGC 209 3747CAGGCUUGAGCCACCACGC 209 3765 GCGUGGUGGCUCAAGCCUG 676 3765CCCGGCUAAAACAUUGCAA 210 3765 CCCGGCUAAAACAUUGCAA 210 3783UUGCAAUGUUUUAGCCGGG 677 3783 AAUUUAAAUGAGAGUUUUA 211 3783AAUUUAAAUGAGAGUUUUA 211 3801 UAAAACUCUCAUUUAAAUU 678 3801AAAAAUUAAAUAAUGACUG 212 3801 AAAAAUUAAAUAAUGACUG 212 3819CAGUCAUUAUUUAAUUUUU 679 3819 GCCCUGUUUCUGUUUUAGU 213 3819GCCCUGUUUCUGUUUUAGU 213 3837 ACUAAAACAGAAACAGGGC 680 3837UAUGUAAAUCCUCAGUUCU 214 3837 UAUGUAAAUCCUCAGUUCU 214 3855AGAACUGAGGAUUUACAUA 681 3855 UUCACCUUUGCACUGUCUG 215 3855UUCACCUUUGCACUGUCUG 215 3873 CAGACAGUGCAAAGGUGAA 682 3873GCCACUUAGUUUGGUUAUA 216 3873 GCCACUUAGUUUGGUUAUA 216 3891UAUAACCAAACUAAGUGGC 683 3891 AUAGUCAUUAACUUGAAUU 217 3891AUAGUCAUUAACUUGAAUU 217 3909 AAUUCAAGUUAAUGACUAU 684 3909UUGGUCUGUAUAGUCUAGA 218 3909 UUGGUCUGUAUAGUCUAGA 218 3927UCUAGACUAUACAGACCAA 685 3927 ACUUUAAAUUUAAAGUUUU 219 3927ACUUUAAAUUUAAAGUUUU 219 3945 AAAACUUUAAAUUUAAAGU 686 3945UCUACAAGGGGAGAAAAGU 220 3945 UCUACAAGGGGAGAAAAGU 220 3963ACUUUUCUCCCCUUGUAGA 687 3963 UGUUAAAAUUUUUAAAAUA 221 3963UGUUAAAAUUUUUAAAAUA 221 3981 UAUUUUAAAAAUUUUAACA 688 3981AUGUUUUCCAGGACACUUC 222 3981 AUGUUUUCCAGGACACUUC 222 3999GAAGUGUCCUGGAAAACAU 689 3999 CACUUCCAAGUCAGGUAGG 223 3999CACUUCCAAGUCAGGUAGG 223 4017 CCUACCUGACUUGGAAGUG 690 4017GUAGUUCAAUCUAGUUGUU 224 4017 GUAGUUCAAUCUAGUUGUU 224 4035AACAACUAGAUUGAACUAC 691 4035 UAGCCAAGGACUCAAGGAC 225 4035UAGCCAAGGACUCAAGGAC 225 4053 GUCCUUGAGUCCUUGGCUA 692 4053CUGAAUUGUUUUAACAUAA 226 4053 CUGAAUUGUUUUAACAUAA 226 4071UUAUGUUAAAACAAUUCAG 693 4071 AGGCUUUUCCUGUUCUGGG 227 4071AGGCUUUUCCUGUUCUGGG 227 4089 CCCAGAACAGGAAAAGCCU 694 4089GAGCCGCACUUCAUUAAAA 228 4089 GAGCCGCACUUCAUUAAAA 228 4107UUUUAAUGAAGUGCGGCUC 695 4107 AUUCUUCUAAAACUUGUAU 229 4107AUUCUUCUAAAACUUGUAU 229 4125 AUACAAGUUUUAGAAGAAU 696 4125UGUUUAGAGUUAAGCAAGA 230 4125 UGUUUAGAGUUAAGCAAGA 230 4143UCUUGCUUAACUCUAAACA 697 4143 ACUUUUUUUCUUCCUCUCC 231 4143ACUUUUUUUCUUCCUCUCC 231 4161 GGAGAGGAAGAAAAAAAGU 698 4161CAUGAGUUGUGAAAUUUAA 232 4161 CAUGAGUUGUGAAAUUUAA 232 4179UUAAAUUUCACAACUCAUG 699 4179 AUGCACAACGCUGAUGUGG 233 4179AUGCACAACGCUGAUGUGG 233 4197 CCACAUCAGCGUUGUGCAU 700 4197GCUAACAAGUUUAUUUUAA 234 4197 GCUAACAAGUUUAUUUUAA 234 4215UUAAAAUAAACUUGUUAGC 701 4215 AGAAUUGUUUAGAAAUGCU 235 4215AGAAUUGUUUAGAAAUGCU 235 4233 AGCAUUUCUAAACAAUUCU 702 4233UGUUGCUUCAGGUUCUUAA 236 4233 UGUUGCUUCAGGUUCUUAA 236 4251UUAAGAACCUGAAGCAACA 703 4251 AAAUCACUCAGCACUCCAA 237 4251AAAUCACUCAGCACUCCAA 237 4269 UUGGAGUGCUGAGUGAUUU 704 4269ACUUCUAAUCAAAUUUUUG 238 4269 ACUUCUAAUCAAAUUUUUG 238 4287CAAAAAUUUGAUUAGAAGU 705 4287 GGAGACUUAACAGCAUUUG 239 4287GGAGACUUAACAGCAUUUG 239 4305 CAAAUGCUGUUAAGUCUCC 706 4305GUCUGUGUUUGAACUAUAA 240 4305 GUCUGUGUUUGAACUAUAA 240 4323UUAUAGUUCAAACACAGAC 707 4323 AAAAGCACCGGAUCUUUUC 241 4323AAAAGCACCGGAUCUUUUC 241 4341 GAAAAGAUCCGGUGCUUUU 708 4341CCAUCUAAUUCCGCAAAAA 242 4341 CCAUCUAAUUCCGCAAAAA 242 4359UUUUUGCGGAAUUAGAUGG 709 4359 AUUGAUCAUUUGCAAAGUC 243 4359AUUGAUCAUUUGCAAAGUC 243 4377 GACUUUGCAAAUGAUCAAU 710 4377CAAAACUAUAGCCAUAUCC 244 4377 CAAAACUAUAGCCAUAUCC 244 4395GGAUAUGGCUAUAGUUUUG 711 4395 CAAAUCUUUUCCCCCUCCC 245 4395CAAAUCUUUUCCCCCUCCC 245 4413 GGGAGGGGGAAAAGAUUUG 712 4413CAAGAGUUCUCAGUGUCUA 246 4413 CAAGAGUUCUCAGUGUCUA 246 4431UAGACACUGAGAACUCUUG 713 4431 ACAUGUAGACUAUUCCUUU 247 4431ACAUGUAGACUAUUCCUUU 247 4449 AAAGGAAUAGUCUACAUGU 714 4449UUCUGUAUAAAGUUCACUC 248 4449 UUCUGUAUAAAGUUCACUC 248 4467GAGUGAACUUUAUACAGAA 715 4467 CUAGGAUUUCAAGUCACCA 249 4467CUAGGAUUUCAAGUCACCA 249 4485 UGGUGACUUGAAAUCCUAG 716 4485ACUUAUUUUACAUUUUAGU 250 4485 ACUUAUUUUACAUUUUAGU 250 4503ACUAAAAUGUAAAAUAAGU 717 4503 UCAUGCAAAGAUUCAAGUA 251 4503UCAUGCAAAGAUUCAAGUA 251 4521 UACUUGAAUCUUUGCAUGA 718 4521AGUUUUGCAAUAAGUACUU 252 4521 AGUUUUGCAAUAAGUACUU 252 4539AAGUACUUAUUGCAAAACU 719 4539 UAUCUUUAUUUGUAAUAAU 253 4539UAUCUUUAUUUGUAAUAAU 253 4557 AUUAUUACAAAUAAAGAUA 720 4557UUUAGUCUGCUGAUCAAAA 254 4557 UUUAGUCUGCUGAUCAAAA 254 4575UUUUGAUCAGCAGACUAAA 721 4575 AGCAUUGUCUUAAUUUUUG 255 4575AGCAUUGUCUUAAUUUUUG 255 4593 CAAAAAUUAAGACAAUGCU 722 4593GAGAACUGGUUUUAGCAUU 256 4593 GAGAACUGGUUUUAGCAUU 256 4611AAUGCUAAAACCAGUUCUC 723 4611 UUACAAACUAAAUUCCAGU 257 4611UUACAAACUAAAUUCCAGU 257 4629 ACUGGAAUUUAGUUUGUAA 724 4629UUAAUUAAUUAAUAGCUUU 258 4629 UUAAUUAAUUAAUAGCUUU 250 4647AAAGCUAUUAAUUAAUUAA 725 4647 UAUAUUGCCUUUCCUGCUA 259 4647UAUAUUGCCUUUCCUGCUA 259 4665 UAGCAGGAAAGGCAAUAUA 726 4665ACAUUUGGUUUUUUCCCCU 260 4665 ACAUUUGGUUUUUUCCCCU 260 4683AGGGGAAAAAACCAAAUGU 727 4683 UGUCCCUUUGAUUACGGGC 261 4683UGUCCCUUUGAUUACGGGC 261 4701 GCCCGUAAUCAAAGGGACA 728 4701CUAAGGUAGGGUAGAGUGG 262 4701 CUAAGGUAGGGUAGAGUGG 262 4719CCACUCUACCCUACCUUAG 729 4719 GGUGUAGUGAGUGUAUAUA 263 4719GGUGUAGUGAGUGUAUAUA 263 4737 UAUAUACACUCACUACACC 730 4737AAUGUGAUUUGGCCCUGUG 264 4737 AAUGUGAUUUGGCCCUGUG 264 4755CACAGGGCCAAAUCACAUU 731 4755 GUAUUAUGAUAUUUUGUUA 265 4755GUAUUAUGAUAUUUUGUUA 265 4773 UAACAAAAUAUCAUAAUAC 732 4773AUUUUUGUUGUUAUAUUAU 266 4773 AUUUUUGUUGUUAUAUUAU 266 4791AUAAUAUAACAACAAAAAU 733 4791 UUUACAUUUCAGUAGUUGU 267 4791UUUACAUUUCAGUAGUUGU 267 4809 ACAACUACUGAAAUGUAAA 734 4809UUUUUUGUGUUUCCAUUUU 268 4809 UUUUUUGUGUUUCCAUUUU 268 4827AAAAUGGAAACACAAAAAA 735 4827 UAGUGGAUAAAAUUUGUAU 269 4827UAGUGGAUAAAAUUUGUAU 269 4845 AUACAAAUUUUAUCCACUA 736 4845UUUUGAACUAUGAAUGGAG 270 4845 UUUUGAACUAUGAAUGGAG 270 4863CUCCAUUCAUAGUUCAAAA 737 4863 GACUACCGCCCCAGCAUUA 271 4863GACUACCGCCCCAGCAUUA 271 4881 UAAUGCUGGGGCGGUAGUC 738 4881AGUUUCACAUGAUAUACCC 272 4881 AGUUUCACAUGAUAUACCC 272 4899GGGUAUAUCAUGUGAAACU 739 4899 CUUUAAACCCGAAUCAUUG 273 4899CUUUAAACCCGAAUCAUUG 273 4917 CAAUGAUUCGGGUUUAAAG 740 4917GUUUUAUUUCCUGAUUACA 274 4917 GUUUUAUUUCCUGAUUACA 274 4935UGUAAUCAGGAAAUAAAAC 741 4935 ACAGGUGUUGAAUGGGGAA 275 4935ACAGGUGUUGAAUGGGGAA 275 4953 UUCCCCAUUCAACACCUGU 742 4953AAGGGGCUAGUAUAUCAGU 276 4953 AAGGGGCUAGUAUAUCAGU 276 4971ACUGAUAUACUAGCCCCUU 743 4971 UAGGAUAUACUAUGGGAUG 277 4971UAGGAUAUACUAUGGGAUG 277 4989 CAUCCCAUAGUAUAUCCUA 744 4989GUAUAUAUAUCAUUGCUGU 278 4989 GUAUAUAUAUCAUUGCUGU 278 5007ACAGCAAUGAUAUAUAUAC 745 5007 UUAGAGAAAUGAAAUAAAA 279 5007UUAGAGAAAUGAAAUAAAA 279 5025 UUUUAUUUCAUUUCUCUAA 746 5025AUGGGGCUGGGCUCAGUGG 280 5025 AUGGGGCUGGGCUCAGUGG 280 5043CCACUGAGCCCAGCCCCAU 747 5043 GCUCACGCCUGUAAUCCCA 281 5043GCUCACGCCUGUAAUCCCA 281 5061 UGGGAUUACAGGCGUGAGC 748 5061AGCACUUUGGGAGGCUGAG 282 5061 AGCACUUUGGGAGGCUGAG 282 5079CUCAGCCUCCCAAAGUGCU 749 5079 GGCAGGUGGAUCACGAGGU 283 5079GGCAGGUGGAUCACGAGGU 283 5097 ACCUCGUGAUCCACCUGCC 750 5097UCAGGAGAUGGAGACCAUC 284 5097 UCAGGAGAUGGAGACCAUC 284 5115GAUGGUCUCGAUCUCCUGA 751 5115 CCUGGCUAACACGGUGAAA 285 5115CCUGGCUAACACGGUGAAA 285 5133 UUUCACCGUGUUAGCCAGG 752 5133ACCCCGUCUCUACUAAAAA 286 5133 ACCCCGUCUCUACUAAAAA 286 5151UUUUUAGUAGAGACGGGGU 753 5151 AACAGAAAAUUAGCCGGGC 287 5151AACAGAAAAUUAGCCGGGC 287 5169 GCCCGGCUAAUUUUCUGUU 754 5169CGUGGUGGCGGGCGCCUGU 288 5169 CGUGGUGGCGGGCGCCUGU 288 5187ACAGGCGCCCGCCACCACG 755 5187 UAGUCCCAGCUACUCGGGA 289 5187UAGUCCCAGCUACUCGGGA 289 5205 UCCCGAGUAGCUGGGACUA 756 5205AGGCUGAGGCAGGAGAAUG 290 5205 AGGCUGAGGCAGGAGAAUG 290 5223CAUUCUCCUGCCUCAGCCU 757 5223 GGUGUGAACCCGGGAGGCA 291 5223GGUGUGAACCCGGGAGGCA 291 5241 UGCCUCCCGGGUUCACACC 758 5241AGAGCUUGCAGUGAGCCGA 292 5241 AGAGCUUGCAGUGAGCCGA 292 5259UCGGCUCACUGCAAGCUCU 759 5259 AGAUCUCGCCACUGCACUC 293 5259AGAUCUCGCCACUGCACUC 293 5277 GAGUGCAGUGGCGAGAUCU 760 5277CCAGCCUGGGCAACAGAGC 294 5277 CCAGCCUGGGCAACAGAGC 294 5295GCUCUGUUGCCCAGGCUGG 761 5295 CAAGACUCUGUCUCAAAAA 295 5295CAAGACUCUGUCUCAAAAA 295 5313 UUUUUGAGACAGAGUCUUG 762 5313AAAAAAAAAAAAGAAAUAA 296 5313 AAAAAAAAAAAAGAAAUAA 296 5331UUAUUUCUUUUUUUUUUUU 763 5331 AGAAAAUGGGAAGCAAUAU 297 5331AGAAAAUGGGAAGCAAUAU 297 5349 AUAUUGCUUCCCAUUUUCU 764 5349UUUGACAUAGUUCUUUUUA 298 5349 UUUGACAUAGUUCUUUUUA 298 5367UAAAAAGAACUAUGUCAAA 765 5367 AGUCAAAUCUACUUGUUAA 299 5367AGUCAAAUCUACUUGUUAA 299 5385 UUAACAAGUAGAUUUGACU 766 5385AAAAAAGGGUAGCAGUUUA 300 5385 AAAAAAGGGUAGCAGUUUA 300 5403UAAACUGCUACCCUUUUUU 767 5403 AUUCAUCUGUGAAAGGAAA 301 5403AUUCAUCUGUGAAAGGAAA 301 5421 UUUCCUUUCACAGAUGAAU 768 5421AAUAAUACUUAUCUUACAA 302 5421 AAUAAUACUUAUCUUACAA 302 5439UUGUAAGAUAAGUAUUAUU 769 5439 AGGUUGCAAGAGCUCAAGG 303 5439AGGUUGCAAGAGCUCAAGG 303 5457 CCUUGAGCUCUUGCAACCU 770 5457GAGACCAUGUAUGUAAAGU 304 5457 GAGACCAUGUAUGUAAAGU 304 5475ACUUUACAUACAUGGUCUC 771 5475 UUCCUGCUGUAAAUAUGAA 305 5475UUCCUGCUGUAAAUAUGAA 305 5493 UUCAUAUUUACAGCAGGAA 772 5493ACUCCCAUCCUAAUACCCU 306 5493 ACUCCCAUCCUAAUACCCU 306 5511AGGGUAUUAGGAUGGGAGU 773 5511 UUUUACCUCUCUGUGGGUU 307 5511UUUUACCUCUCUGUGGGUU 307 5529 AACCCACAGAGAGGUAAAA 774 5529UUGUCUUGACCUGGAAAUU 308 5529 UUGUCUUGACCUGGAAAUU 308 5547AAUUUCCAGGUCAAGACAA 775 5547 UUGGGCUAAAACUUAGAAA 309 5547UUGGGCUAAAACUUAGAAA 309 5565 UUUCUAAGUUUUAGCCCAA 776 5565AAAAUUCUUACAUGAUAAC 310 5565 AAAAUUCUUACAUGAUAAC 310 5583GUUAUCAUGUAAGAAUUUU 777 5583 CUCAGUGAUGCUUACUCAU 311 5583CUCAGUGAUGCUUACUCAU 311 5601 AUGAGUAAGCAUCACUGAG 778 5601UAGUUUUUGGUGUUUCUCA 312 5601 UAGUUUUUGGUGUUUCUCA 312 5619UGAGAAACACCAAAAACUA 779 5619 AUAGAUAAGAUAUAAAUCA 313 5619AUAGAUAAGAUAUAAAUCA 313 5637 UGAUUUAUAUCUUAUCUAU 780 5637AGCUGGGCGCGGUGGCUCA 314 5637 AGCUGGGCGCGGUGGCUCA 314 5655UGAGCCACCGCGCCCAGCU 781 5655 AUGCCUGUAAUCCCAGCAC 315 5655AUGCCUGUAAUCCCAGCAC 315 5673 GUGCUGGGAUUACAGGCAU 782 5673CUUUGGGAGGCCGAGGCGG 316 5673 CUUUGGGAGGCCGAGGCGG 316 5691CCGCCUCGGCCUCCCAAAG 783 5691 GGCAGAUCACCUGAGGUCG 317 5691GGCAGAUCACCUGAGGUCG 317 5709 CGACCUCAGGUGAUCUGCC 784 5709GGGAGGUCGAGACCAGCCU 318 5709 GGGAGGUCGAGACCAGCCU 318 5727AGGCUGGUCUCGACCUCCC 785 5727 UGACCAACAUGGAGAAACC 319 5727UGACCAACAUGGAGAAACC 319 5745 GGUUUCUCCAUGUUGGUCA 786 5745CCCGUCUCUACUAAAAAUA 320 5745 CCCGUCUCUACUAAAAAUA 320 5763UAUUUUUAGUAGAGACGGG 787 5763 ACAAAAUUAGCUGGGCGUG 321 5763ACAAAAUUAGCUGGGCGUG 321 5781 CACGCCCAGCUAAUUUUGU 788 5781GGUGGCUCAUGCCUGUAAU 322 5781 GGUGGCUCAUGCCUGUAAU 322 5799AUUACAGGCAUGAGCCACC 789 5799 UCCCAGCUACUUGGGAGGC 323 5799UCCCAGCUACUUGGGAGGC 323 5817 GCCUCCCAAGUAGCUGGGA 790 5817CUGAGGCAGGAGAAUCGCU 324 5817 CUGAGGCAGGAGAAUCGCU 324 5835AGCGAUUCUCCUGCCUCAG 791 5835 UUGAACCCAGGAGGCGGAG 325 5835UUGAACCCAGGAGGCGGAG 325 5853 CUCCGCCUCCUGGGUUCAA 792 5853GGUUGUGGUGAGCGAAGAU 326 5853 GGUUGUGGUGAGCGAAGAU 326 5871AUCUUCGCUCACCACAACC 793 5871 UCGUGCCAUUGCACUCCAG 327 5871UCGUGCCAUUGCACUCCAG 327 5889 CUGGAGUGCAAUGGCACGA 794 5889GCCUGGGCAACAAGAGCAA 328 5889 GCCUGGGCAACAAGAGCAA 328 5907UUGCUCUUGUUGCCCAGGC 795 5907 AAACUCUGUCUCAAAAAAA 329 5907AAACUCUGUCUCAAAAAAA 329 5925 UUUUUUUGAGACAGAGUUU 796 5925AAAAAAAGAUAUAAAUCAC 330 5925 AAAAAAAGAUAUAAAUCAC 330 5943GUGAUUUAUAUCUUUUUUU 797 5943 CAAUAAAUAAAUAGGUCAA 331 5943CAAUAAAUAAAUAGGUCAA 331 5961 UUGACCUAUUUAUUUAUUG 798 5961AUACAAAUGUUAGCCAGGC 332 5961 AUACAAAUGUUAGCCAGGC 332 5979GCCUGGCUAACAUUUGUAU 799 5979 CGUGGUGGCACAUGCCCAU 333 5979CGUGGUGGCACAUGCCCAU 333 5997 AUGGGCAUGUGCCACCACG 800 5997UAGUCGCAGCUACUCUGGA 334 5997 UAGUCGCAGCUACUCUGGA 334 6015UCCAGAGUAGCUGCGACUA 801 6015 AGGCAGAGGCAGGAGGAUC 335 6015AGGCAGAGGCAGGAGGAUC 335 6033 GAUCCUCCUGCCUCUGCCU 802 6033CACUUGAGCCCAUGAAUUU 336 6033 CACUUGAGCCCAUGAAUUU 336 6051AAAUUCAUGGGCUCAAGUG 803 6051 UGAGGCAGCAGUGAGCUAU 337 6051UGAGGCAGCAGUGAGCUAU 337 6069 AUAGCUCACUGCUGCCUCA 804 6069UGAUUGUGCCACUGUACUC 338 6069 UGAUUGUGCCACUGUACUC 338 6087GAGUACAGUGGCACAAUCA 805 6087 CCAGUCUGGGUGACAGAGU 339 6087CCAGUCUGGGUGACAGAGU 339 6105 ACUCUGUCACCCAGACUGG 806 6105UGAGACCCCAUCUCUAAAU 340 6105 UGAGACCCCAUCUCUAAAU 340 6123AUUUAGAGAUGGGGUCUCA 807 6123 UAAAUAGGUCAAACCCUUA 341 6123UAAAUAGGUCAAACCCUUA 341 6141 UAAGGGUUUGACCUAUUUA 808 6141AAAAAUAUUUAAAUUCUUA 342 6141 AAAAAUAUUUAAAUUCUUA 342 6159UAAGAAUUUAAAUAUUUUU 809 6159 AAAAAAUUGAAAAGAUUAU 343 6159AAAAAAUUGAAAAGAUUAU 343 6177 AUAAUCUUUUCAAUUUUUU 810 6177UUCUUCUCAAAUUUAGUUG 344 6177 UUCUUCUCAAAUUUAGUUG 344 6195CAACUAAAUUUGAGAAGAA 811 6195 GAGCUUUCUAAGAGAAGCA 345 6195GAGCUUUCUAAGAGAAGCA 345 6213 UGCUUCUCUUAGAAAGCUC 812 6213AAUUGGCUUUUUCCCACUU 346 6213 AAUUGGCUUUUUCCCACUU 346 6231AAGUGGGAAAAAGCCAAUU 813 6231 UCAAUAAUCAUUUUCAGUU 347 6231UCAAUAAUCAUUUUCAGUU 347 6249 AACUGAAAAUGAUUAUUGA 814 6249UUGACUCAUACAGUUAACA 348 6249 UUGACUCAUACAGUUAACA 348 6267UGUUAACUGUAUGAGUCAA 815 6267 ACAAUGUGAAUUUCUUCCU 349 6267ACAAUGUGAAUUUCUUCCU 349 6285 AGGAAGAAAUUCACAUUGU 816 6285UCAGCAUAACAGAGUUAUA 350 6285 UCAGCAUAACAGAGUUAUA 350 6303UAUAACUCUGUUAUGCUGA 817 6303 AGAAUGACAGGGCUGGAAG 351 6303AGAAUGACAGGGCUGGAAG 351 6321 CUUCCAGCCCUGUCAUUCU 818 6321GUGACCUUAGAGAGUAUCC 352 6321 GUGACCUUAGAGAGUAUCC 352 6339GGAUACUCUCUAAGGUCAC 819 6339 CAGUUCUUUCAUUUUACAG 353 6339CAGUUCUUUCAUUUUACAG 353 6357 CUGUAAAAUGAAAGAACUG 820 6357GGUGAGGCAACUGAGACUC 354 6357 GGUGAGGCAACUGAGACUC 354 6375GAGUCUCAGUUGCCUCACC 821 6375 CAAAGGUGAUGUAAUUUGU 355 6375CAAAGGUGAUGUAAUUUGU 355 6393 ACAAAUUACAUCACCUUUG 822 6393UGCAAAGAUUAUAGCUAAU 356 6393 UGCAAAGAUUAUAGCUAAU 356 6411AUUAGCUAUAAUCUUUGCA 823 6411 UUAGUAGCAGAGCCCUGAC 357 6411UUAGUAGCAGAGCCCUGAC 357 6429 GUCAGGGCUCUGCUACUAA 824 6429CUGGGACAUAGUUUGAAGG 358 6429 CUGGGACAUAGUUUGAAGG 358 6447CCUUCAAACUAUGUCCCAG 825 6447 GUGAAAAACUUCACCAAGC 359 6447GUGAAAAACUUCACCAAGC 359 6465 GCUUGGUGAAGUUUUUCAC 826 6465CUACCUUUCUUGAAAGGUC 360 6465 CUACCUUUCUUGAAAGGUC 360 6483GACCUUUCAAGAAAGGUAG 827 6483 CCAAAUGUUUAUGUUUUCA 361 6483CCAAAUGUUUAUGUUUUCA 361 6501 UGAAAACAUAAACAUUUGG 828 6501AACUACUCUUUCCACUGUA 362 6501 AACUACUCUUUCCACUGUA 362 6519UACAGUGGAAAGAGUAGUU 829 6519 ACCAUAACUUUCACUACAU 363 6519ACCAUAACUUUCACUACAU 363 6537 AUGUAGUGAAAGUUAUGGU 830 6537UAUUAAAUGACACUUUAUA 364 6537 UAUUAAAUGACACUUUAUA 364 6555UAUAAAGUGUCAUUUAAUA 831 6555 AACUAAUAUAAUAGGACAA 365 6555AACUAAUAUAAUAGGACAA 365 6573 UUGUCCUAUUAUAUUAGUU 832 6573AUCAUCAAUGCAUAUAUAG 366 6573 AUCAUCAAUGCAUAUAUAG 366 6591CUAUAUAUGCAUUGAUGAU 833 6591 GCCAGCCCUUCAUAUCUGU 367 6591GCCAGCCCUUCAUAUCUGU 367 6609 ACAGAUAUGAAGGGCUGGC 834 6609UGGGUUUUGCAUCCAUGGA 368 6609 UGGGUUUUGCAUCCAUGGA 368 6627UCCAUGGAUGCAAAACCCA 835 6627 AUUCAACCAAGGAGGAAUU 369 6627AUUCAACCAAGGAGGAAUU 369 6645 AAUUCCUCCUUGGUUGAAU 836 6645UGAAAACACUGAGAAAAAA 370 6645 UGAAAACACUGAGAAAAAA 370 6663UUUUUUCUCAGUGUUUUCA 837 6663 AAAAAAAGACCACACAAUA 371 6663AAAAAAAGACCACACAAUA 371 6681 UAUUGUGUGGUCUUUUUUU 838 6681AAAAAAAAAAAAUACAAAA 372 6681 AAAAAAAAAAAAUACAAAA 372 6699UUUUGUAUUUUUUUUUUUU 839 6699 AUAAUACAAAGAAAAAGCC 373 6699AUAAUACAAAGAAAAAGCC 373 6717 GGCUUUUUCUUUGUAUUAU 840 6717CAAAAUUGUCAUACUGUUG 374 6717 CAAAAUUGUCAUACUGUUG 374 6735CAACAGUAUGACAAUUUUG 841 6735 GUUAAGCAACAGUAUAACA 375 6735GUUAAGCAACAGUAUAACA 375 6753 UGUUAUACUGUUGCUUAAC 842 6753AACUAUUUACAUAGCAUUA 376 6753 AACUAUUUACAUAGCAUUA 376 6771UAAUGCUAUGUAAAUAGUU 843 6771 AAGGUUGGUGCAAAAAUGC 377 6771AAGGUUGGUGCAAAAAUGC 377 6789 GCAUUUUUGCACCAACCUU 844 6789CAAAAAAAAAAAAAGCAAU 378 6789 CAAAAAAAAAAAAAGCAAU 378 6807AUUGCUUUUUUUUUUUUUG 845 6807 UUAUUUUUAAACCAACCUA 379 6807UUAUUUUUAAACCAACCUA 379 6825 UAGGUUGGUUUAAAAAUAA 846 6825AAUAUAUUGUAUUAGGUAU 380 6825 AAUAUAUUGUAUUAGGUAU 380 6843AUACCUAAUACAAUAUAUU 847 6843 UUAAAGUCAUCUGGACAUG 381 6843UUAAAGUCAUCUGGACAUG 381 6861 CAUGUCCAGAUGACUUUAA 848 6861GAAUUAAAGUAUAUGAUGC 382 6861 GAAUUAAAGUAUAUGAUGC 382 6879GCAUCAUAUACUUUAAUUC 849 6879 CCAGCCUGGACAAAAGGCA 383 6879CCAGCCUGGACAAAAGGCA 383 6897 UGCCUUUUGUCCAGGCUGG 850 6897AAAACCCUGUCUCUACAAA 384 6897 AAAACCCUGUCUCUACAAA 384 6915UUUGUAGAGACAGGGUUUU 851 6915 AAAAUACAAAAAUUAGCUG 385 6915AAAAUACAAAAAUUAGCUG 385 6933 CAGCUAAUUUUUGUAUUUU 852 6933GGGCAUGGUGGUGUGUGCC 386 6933 GGGCAUGGUGGUGUGUGCC 386 6951GGCACACACCACCAUGCCC 853 6951 CUGUAGUCCUGGCUACUCC 387 6951CUGUAGUCCUGGCUACUCC 387 6969 GGAGUAGCCAGGACUACAG 854 6969CGGAGCCUGAGGUGGGAGG 388 6969 CGGAGCCUGAGGUGGGAGG 388 6987CCUCCCACCUCAGGCUCCG 855 6987 GAUCGCUUGAGUCUGGGAG 389 6987GAUCGCUUGAGUCUGGGAG 389 7005 CUCCCAGACUCAAGCGAUC 856 7005GGCAGAGGCUGCAUUGAGC 390 7005 GGCAGAGGCUGCAUUGAGC 390 7023GCUCAAUGCAGCCUCUGCC 857 7023 CUAUGAUCAUGGCACUGCA 391 7023CUAUGAUCAUGGCACUGCA 391 7041 UGCAGUGCCAUGAUCAUAG 858 7041AUUCCAGCCUGGGUGACAG 392 7041 AUUCCAGCCUGGGUGACAG 392 7059CUGUCACCCAGGCUGGAAU 859 7059 GUGCAAGACCUUGUCUCAG 393 7059GUGCAAGACCUUGUCUCAG 393 7077 CUGAGACAAGGUCUUGCAC 860 7077GAAUAAAUAAAGUAUGUGA 394 7077 GAAUAAAUAAAGUAUGUGA 394 7095UCACAUACUUUAUUUAUUC 861 7095 AUGAAGAUGUGCAUACAUU 395 7095AUGAAGAUGUGCAUACAUU 395 7113 AAUGUAUGCACAUCUUCAU 862 7113UAUAUGCAAAUACUGUUUU 396 7113 UAUAUGCAAAUACUGUUUU 396 7131AAAACAGUAUUUGCAUAUA 863 7131 UUUUUUUUUUUAAUUUAAA 397 7131UUUUUUUUUUUAAUUUAAA 397 7149 UUUAAAUUAAAAAAAAAAA 864 7149ACAGUCUCACUGUGUUGCC 398 7149 ACAGUCUCACUGUGUUGCC 398 7167GGCAACACAGUGAGACUGU 865 7167 CCAGGAUGGAGUGCAAUGG 399 7167CCAGGAUGGAGUGCAAUGG 399 7185 CCAUUGCACUCCAUCCUGG 866 7185GCACAAUCUUGGCUCAUGG 400 7185 GCACAAUCUUGGCUCAUGG 400 7203CCAUGAGCCAAGAUUGUGC 867 7203 GCAAACUCUGCCUCGCAAG 401 7203GCAAACUCUGCCUCGCAAG 401 7221 CUUGCGAGGCAGAGUUUGC 868 7221GCAGCUGGGACUACAGGCA 402 7221 GCAGCUGGGACUACAGGCA 402 7239UGCCUGUAGUCCCAGCUGC 869 7239 AUGCUCCACGGUGCCCAGU 403 7239AUGCUCCACGGUGCCCAGU 403 7257 ACUGGGCACCGUGGAGCAU 870 7257UUAAUUUUUUUUGUAUUCU 404 7257 UUAAUUUUUUUUGUAUUCU 404 7275AGAAUACAAAAAAAAUUAA 871 7275 UUAGUAGAGACAGGGUUUC 405 7275UUAGUAGAGACAGGGUUUC 405 7293 GAAACCGUCUCUCUACUAA 872 7293CACCAUGUUGGCCAGGCUA 406 7293 CACCAUGUUGGCCAGGCUA 406 7311UAGCCUGGCCAACAUGGUG 873 7311 AGUCUUGAAUUUCUGACCU 407 7311AGUCUUGAAUUUCUGACCU 407 7329 AGGUCAGAAAUUCAAGACU 874 7329UCAAGUGAUUCAUCUCCCA 408 7329 UCAAGUGAUUCAUCUCCCA 408 7347UGGGAGAUGAAUCACUUGA 875 7347 AAAGUGCUGGGAUUACAGG 409 7347AAAGUGCUGGGAUUACAGG 409 7365 CCUGUAAUCCCAGCACUUU 876 7365GCGUGAGCCACCACGGCCG 410 7365 GCGUGAGCCACCACGGCCG 410 7383CGGCCGUGGUGGCUCACGC 877 7383 GGCUAAUUUUUGUAUUUUU 411 7383GGCUAAUUUUUGUAUUUUU 411 7401 AAAAAUACAAAAAUUAGCC 878 7401UUAGUAGUGACUGGUUUCG 412 7401 UUAGUAGUGACUGGUUUCG 412 7419CGAAACCAGUCACUACUAA 879 7419 GCGGUGUUGACCAGGCUGG 413 7419GCGGUGUUGACCAGGCUGG 413 7437 CCAGCCUGGUCAACACCGC 880 7437GUCUCGAACUCCUGAUCUC 414 7437 GUCUCGAACUCCUGAUCUC 414 7455GAGAUCAGGAGUUCGAGAC 881 7455 CAGGUGAUCUGCCUGCCUC 415 7455CAGGUGAUCUGCCUGCCUC 415 7473 GAGGCAGGCAGAUCACCUG 882 7473CGGCCUCACAAAGUGCUGG 416 7473 CGGCCUCACAAAGUGCUGG 416 7491CCAGCACUUUGUGAGGCCG 883 7491 GGAUUACAGGUGUGAACCA 417 7491GGAUUACAGGUGUGAACCA 417 7509 UGGUUCACACCUGUAAUCC 884 7509ACUGCUCCCGGCCUUGUGU 418 7509 ACUGCUCCCGGCCUUGUGU 418 7527ACACAAGGCCGGGAGCAGU 885 7527 UGAUUUUAUCUAAGGGACU 419 7527UGAUUUUAUCUAAGGGACU 419 7545 AGUCCCUUAGAUAAAAUCA 886 7545UUAAGCGUCCUCAGGUCCU 420 7545 UUAAGCGUCCUCAGGUCCU 420 7563AGGACCUGAGGACGCUUAA 887 7563 UAGGGGGUCGUGAAACCAA 421 7563UAGGGGGUCGUGAAACCAA 421 7581 UUGGUUUCACGACCCCCUA 888 7581AAACCCCAGGGAUAGCAAG 422 7581 AAACCCCAGGGAUAGCAAG 422 7599CUUGCUAUCCCUGGGGUUU 889 7599 GGGACAAUUGUAUCUUCAA 423 7599GGGACAAUUGUAUCUUCAA 423 7617 UUGAAGAUACAAUUGUCCC 890 7617AAGUAGACAAAUGGCGCCG 424 7617 AAGUAGACAAAUGGCGCCG 424 7635CGGCGCCAUUUGUCUACUU 891 7635 GGGCACGGUGGCUCACGCC 425 7635GGGCACGGUGGCUCACGCC 425 7653 GGCGUGAGCCACCGUGCCC 892 7653CUGUAAUCCCAGCAGUUUC 426 7653 CUGUAAUCCCAGCAGUUUC 426 7671GAAACUGCUGGGAUUACAG 893 7671 CCGAGGCUGAGGCAGGCGG 427 7671CCGAGGCUGAGGCAGGCGG 427 7689 CCGCCUGCCUCAGCCUCGG 894 7689GCUCACCUGAGGUCAGGAG 428 7689 GCUCACCUGAGGUCAGGAG 428 7707CUCCUGACCUCAGGUGAGC 895 7707 GUUGGAGACCAGCCUGGCC 429 7707GUUGGAGACCAGCCUGGCC 429 7725 GGCCAGGCUGGUCUCCAAC 896 7725CAACAUGCUGAAACCCUGU 430 7725 CAACAUGCUGAAACCCUGU 430 7743ACAGGGUUUCAGCAUGUUG 897 7743 UCUGUACAAAAAUACAAAA 431 7743UCUGUACAAAAAUACAAAA 431 7761 UUUUGUAUUUUUGUACAGA 898 7761AAUAGCUGGGCAUGGUGGC 432 7761 AAUAGCUGGGCAUGGUGGC 432 7779GCCACCAUGCCCAGCUAUU 899 7779 CGCAUGCCUGUAGUCCCAG 433 7779CGCAUGCCUGUAGUCCCAG 433 7797 CUGGGACUACAGGCAUGCG 900 7797GCUACUAGAGCGACUGAGG 434 7797 GCUACUAGAGCGACUGAGG 434 7815CCUCAGUCGCUCUAGUAGC 901 7815 GCAGGAGAAUUGCUUGAAC 435 7815GCAGGAGAAUUGCUUGAAC 435 7833 GUUCAAGCAAUUCUCCUGC 902 7833CCUGGGAGGCGGAGGUUGC 436 7833 CCUGGGAGGCGGAGGUUGC 436 7851GCAACCUCCGCCUCCCAGG 903 7851 CAGGGAGCCAAGAUGGCGC 437 7851CAGGGAGCCAAGAUGGCGC 437 7869 GCGCCAUCUUGGCUCCCUG 904 7869CCACCGCACUCCAGCCUAG 438 7869 CCACCGCACUCCAGCCUAG 438 7887CUAGGCUGGAGUGCGGUGG 905 7887 GGUGAUAGAGUGAGACUCC 439 7887GGUGAUAGAGUGAGACUCC 439 7905 GGAGUCUCACUCUAUCACC 906 7905CCUCUCAAAAACAAAACAA 440 7905 CCUCUCAAAAACAAAACAA 440 7923UUGUUUUGUUUUUGAGAGG 907 7923 AAACAAAAAAAUUAGACAA 441 7923AAACAAAAAAAUUAGACAA 441 7941 UUGUCUAAUUUUUUUGUUU 908 7941AAUGCUACAUUAAUGUUUG 442 7941 AAUGCUACAUUAAUGUUUG 442 7959CAAACAUUAAUGUAGCAUU 909 7959 GGGUGGUCAGAUUCUACUU 443 7959GGGUGGUCAGAUUCUACUU 443 7977 AAGUAGAAUCUGACCACCC 910 7977UUGAAUCUGAAGUUUGCAG 444 7977 UUGAAUCUGAAGUUUGCAG 444 7995CUGCAAACUUCAGAUUCAA 911 7995 GAUAUGCCUAUAGAUUUUU 445 7995GAUAUGCCUAUAGAUUUUU 445 8013 AAAAAUCUAUAGGCAUAUC 912 8013UGGAGUUUACCACUUUCUU 446 8013 UGGAGUUUACCACUUUCUU 446 8031AAGAAAGUGGUAAACUCCA 913 8031 UAUUCUGUAUCAUUAAUGU 447 8031UAUUCUGUAUCAUUAAUGU 447 8049 ACAUUAAUGAUACAGAAUA 914 8049UAAUAUUUUAAAUUACUAU 448 8049 UAAUAUUUUAAAUUACUAU 448 8067AUAGUAAUUUAAAAUAUUA 915 8067 UAUAUGUUACCAUUUUUCU 449 8067UAUAUGUUACCAUUUUUCU 449 8085 AGAAAAAUGGUAACAUAUA 916 8085UGGAUUUAGUAAGAAAUUU 450 8085 UGGAUUUAGUAAGAAAUUU 450 8103AAAUUUCUUACUAAAUCCA 917 8103 UGCAGUUUUGGUUUGAUGU 451 8103UGCAGUUUUGGUUUGAUGU 451 8121 ACAUCAAACCAAAACUGCA 918 8121UAACAAGGGUUUUAAUGUA 452 8121 UAACAAGGGUUUUAAUGUA 452 8139UACAUUAAAACCCUUGUUA 919 8139 AAUUUAUGUUAGAUUUUGC 453 8139AAUUUAUGUUAGAUUUUGC 453 8157 GCAAAAUCUAACAUAAAUU 920 8157CAUUUUUUUCAUUACUGUU 454 8157 CAUUUUUUUCAUUACUGUU 454 8175AACAGUAAUGAAAAAAAUG 921 8175 UAUAUUUUAACCUGACUGA 455 8175UAUAUUUUAACCUGACUGA 455 8193 UCAGUCAGGUUAAAAUAUA 922 8193ACUGAUCUAAUUGUAUUAG 456 8193 ACUGAUCUAAUUGUAUUAG 456 8211CUAAUACAAUUAGAUCAGU 923 8211 GUAUUGUGAAUAAUCAUGU 457 8211GUAUUGUGAAUAAUCAUGU 457 8229 ACAUGAUUAUUCACAAUAC 924 8229UGAAAUGUUUUGAGACAGA 458 8229 UGAAAUGUUUUGAGACAGA 458 8247UCUGUCUCAAAACAUUUCA 925 8247 AGUACUAUAUUUGUGAAUA 459 8247AGUACUAUAUUUGUGAAUA 459 8265 UAUUCACAAAUAUAGUACU 926 8265AUAAUUUUAUGGUUUUUUU 460 8265 AUAAUUUUAUGGUUUUUUU 460 8283AAAAAAACCAUAAAAUUAU 927 8283 UCACUUAGAACCUUUCUGU 461 8283UCACUUAGAACCUUUCUGU 461 8301 ACAGAAAGGUUCUAAGUGA 928 8301UGUGGAAAACUAAGAAAAU 462 8301 UGUGGAAAACUAAGAAAAU 462 8319AUUUUCUUAGUUUUCCACA 929 8319 UUGCUUUCUGCUGUAUAAU 463 8319UUGCUUUCUGCUGUAUAAU 463 8337 AUUAUACAGCAGAAAGCAA 930 8337UCUGGCAUUCAUUGUAGAU 464 8337 UCUGGCAUUCAUUGUAGAU 464 8355AUCUACAAUGAAUGCCAGA 931 8355 UUAAAGCUUAUUUUUCUGU 465 8355UUAAAGCUUAUUUUUCUGU 465 8373 ACAGAAAAAUAAGCUUUAA 932 8373UGAAUAAAACGUAUUCAAU 466 8373 UGAAUAAAACGUAUUCAAU 466 8391AUUGAAUACGUUUUAUUCA 933 8391 UAAAAUACUAUUCUUUAAA 467 8391UAAAAUACUAUUCUUUAAA 467 8409 UUUAAAGAAUAGUAUUUUA 934 The 3′-ends of theUpper sequence and the Lower sequence of the siNA construct can includean overhang sequence, for example about 1, 2, 3, or 4 nucleotides inlength, preferably 2 nucleotides in length, wherein the overhangingsequence of the lower sequence is optionally complementary to a portionof the target sequence. The upper and lower sequences in the Table canfurther comprise a chemical modification having Formulae I-VII, such asexemplary siNA constructs shown in FIGS. 4 and 5, or havingmodifications described in Table IV or any combination thereof . . .

TABLE III XIAP/BIRC4 Synthetic Modified siNA Constructs Target Seq CmpdSeq Pos Target ID # Aliases Sequence ID  94 GCGAAAAGGUGGACAAGUCCUAU 935BIRC4:96U21 sense siNA GAAAAGGUGGACAAGUCCUTT 943 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:316U21 sense siNAGCUUUAGUUGUCAUGCAGCTT 944 353 UGGAGACUCAGCAGUUGGAAGAC 937 BIRC4:355U21sense siNA GAGACUCAGCAGUUGGAAGTT 945 358 ACUCAGCAGUUGGAAGACACAGG 938BIRC4:360U21 sense siNA UCAGCAGUUGGAAGACACATT 946 429AGUGCCACGCAGUCUACAAAUUC 939 BIRC4:431U21 sense siNAUGCCACGCAGUCUACAAAUTT 947 592 CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:594U21sense siNA AGGAACCCUGGCAUGUAUATT 948 667 CCCCAAGAGAGUUAGCAAGUGCU 941BIRC4:669U21 sense siNA CCAAGAGAGUUAGCAAGUGTT 949 1345CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1347U21 sense siNACUUGAGGUUCUGGUUGCAGTT 950  94 GCGAAAAGGUGGACAAGUCCUAU 935 BIRC4:114L21antisense siNA AGGACUUGUCCACCUUUUCTT 951 (96C) 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:334L21 antisense siNAGCUGCAUGACAACUAAAGCTT 952 (316C) 353 UGGAGACUCAGCAGUUGGAAGAC 937BIRC4:373L21 antisense siNA CUUCCAACUGCUGAGUCUCTT 953 (355C) 358ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:378L21 antisense siNAUGUGUCUUCCAACUGCUGATT 954 (360C) 429 AGUGCCACGCAGUCUACAAAUUC 939BIRC4:449L21 antisense siNA AUUUGUAGACUGCGUGGCATT 955 (431C) 592CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:612L21 antisense siNAUAUACAUGGCAGGGUUCCUTT 956 (594C) 667 CCCCAAGAGAGUUAGCAAGUGCU 941BIRC4:687L21 antisense siNA CACUUGCUAACUCUCUUGGTT 957 (669C) 1345CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1365L21 antisense siNACUGCAACCAGAACCUCAAGTT 958 (1347C)  94 GCGAAAAGGUGGACAAGUCCUAU 935BIRC4:96U21 sense siNA stab04 B GAAAAGGuGGAcAAGuccuTT B 959 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:316U21 sense siNA stab04 BGcuuuAGuuGucAuGcAGcTT B 960 353 UGGAGACUCAGCAGUUGGAAGAC 937 BIRC4:355U21sense siNA stab04 B GAGAcucAGcAGuuGGAAGTT B 961 358ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:360U21 sense siNA stab04 BucAGcAGuuGGAAGAcAcATT B 962 429 AGUGCCACGCAGUCUAGAAAUUC 939 BIRC4:431U21sense siNA stab04 B uGccAcGcAGucuAcAAAuTT B 963 592CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:594U21 sense siNA stab04 BAGGAAcccuGccAuGuAuATT B 964 667 CCCCAAGAGAGUUAGCAAGUGCU 941 BIRC4:669U21sense siNA stab04 B ccAAGAGAGuuAGcAAGuGTT B 965 1345CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1347U21 sense siNA stab04 BcuuGAGGuucuGGuuGcAGTT B 966  94 GCGAAAAGGUGGACAAGUCCUAU 935 BIRC4:114121antisense siNA AGGAcuuGuccAccuuuucTsT 967 (96C) stab05 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:334L21 antisense siNAGcuGcAuGAcAAcuAAAGcTsT 968 (316C) stab05 353 UGGAGACUCAGCAGUUGGAAGAC 937BIRC4:373L21 antisense siNA cuuccAAcuGcuGAGucucTsT 969 (355C) stab05 358ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:378L21 antisense siNAuGuGucuuccAAcuGcuGATsT 970 (360C) stab05 429 AGUGCCACGCAGUCUACAAAUUC 939BIRC4:449L21 antisense siNA AuuuGuAGAcuGcGuGGcATsT 971 (431C) stab05 592CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:612L21 antisense siNAuAuAcAuGGcAGGGuuccuTsT 972 (594C) stab05 667 CCCCAAGAGAGUUAGCAAGUGCU 941BIRC4:687L21 antisense siNA cAcuuGcuAAcucucuuGGTsT 973 (669C) stab051345 CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1365L21 antisense siNAcuGcAAccAGAAccucAAGTsT 974 (1347C) stab05  94 GCGAAAAGGUGGACAAGUCCUAU935 BIRC4:96U21 sense siNA stab07 B GAAAAGGuGGAcAAGuccuTT B 975 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:316U21 sense siNA stab07 BGcuuuAGuuGucAuGcAGcTT B 976 353 UGGAGACUCAGCAGUUGGAAGAC 937 BIRC4:355U21sense siNA stab07 B GAGAcucAGcAGuuGGAAGTT B 977 358ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:360U21 sense siNA stab07 BucAGcAGuuGGAAGAcAcATT B 978 429 AGUGCCACGCAGUCUACAAAUUC 939 BIRC4:431U21sense siNA stab07 B uGccAcGcAGucuAcAAAuTT B 979 592CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:594U21 sense siNA stab07 BAGGAAcccuGccAuGuAuATT B 980 667 CCCCAAGAGAGUUAGCAAGUGCU 941 BIRC4:669U21sense siNA stab07 B ccAAGAGAGuuAGcAAGuGTT B 981 1345CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1347U21 sense siNA stab07 BcuuGAGGuucuGGuuGcAGTT B 982  94 GCGAAAAGGUGGACAAGUCCUAU 935 BIRC4:114L21antisense siNA AGGAcuuGuccAccuuuucTsT 983 (96C) stab11 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:334L21 antisense siNAGcuGcAuGAcAAcuAAAGcTsT 984 (316C) stab11 353 UGGAGACUCAGCAGUUGGAAGAC 937BIRC4:373L21 antisense siNA cuuccAAcuGcuGAGucucTsT 985 (355C) stab11 358ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:378L21 antisense siNAuGuGucuuccAAcuGcuGATsT 986 (360C) stab11 429 AGUGCCACGCAGUCUACAAAUUC 939BIRC4:449L21 antisense siNA AuuuGuAGAcuGcGuGGcATsT 987 (431C) stab11 592CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:612L21 antisense siNAuAuAcAuGGcAGGGuuccuTsT 988 (594C) stab11 667 CCCCAAGAGAGUUAGCAAGUGCU 941BIRC4:687L21 antisense siNA cAcuuGcuAAcucucuuGGTsT 989 (669C) stab111345 CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1365L21 antisense siNAcuGcAAccAGAAccucAAGTsT 990 (1347C) stab11  94 GCGAAAAGGUGGACAAGUCCUAU935 BIRC4:96U21 sense siNA stab18 B GAAAAGGuGGAcAAGuccuTT B 991 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:316U21 sense siNA stab18 BGcuuuAGuuGucAuGcAGcTT B 992 353 UGGAGACUCAGCAGUUGGAAGAC 937 BIRC4:355U21sense siNA stab18 B GAGAcucAGcAGuuGGAAGTT B 993 358ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:360U21 sense siNA stab18 BucAGcAGuuGGAAGAcAcATT B 994 429 AGUGCCACGCAGUCUACAAAUUC 939 BIRC4:431U21sense siNA stab18 B uGccAcGcAGucuAcAAAuTT B 995 592CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:594U21 sense siNA stab18 BAGGAAcccuGccAuGuAuATT B 996 667 CCCCAAGAGAGUUAGCAAGUGCU 941 BIRC4:669U21sense siNA stab18 B ccAAGAGAGuuAGcAAGuGTT B 997 1345CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1347U21 sense siNA stab18 BcuuGAGGuucuGGuuGcAGTT B 998  94 GCGAAAAGGUGGACAAGUCCUAU 935 33645BIRC4:114121 antisense siNA AGGAcuuGuccAccuuuucTsT 999 (96C) stab08 314GUGCUUUAGUUGUCAUGCAGCUG 936 33646 BIRC4:334L21 antisense siNAGcuGcAuGAcAAcuAAAGcTsT 1000 (316C) stab08 353 UGGAGACUCAGCAGUUGGAAGAC937 33647 BIRG4:373L21 antisense siNA cuuccAAcuGcuGAGucucTsT 1001 (355C)stab08 358 ACUCAGCAGUUGGAAGACACAGG 938 33648 BIRC4:378L21 antisense siNAuGuGucuuccAAcuGcuGATsT 1002 (360C) stab08 429 AGUGCCACGCAGUCUACAAAUUC939 33649 BIRC4:449L21 antisense siNA AuuuGuAGAcuGcGuGGcATsT 1003 (431C)stab08 592 CGAGGAACCCUGCCAUGUAUAGU 940 33650 BIRC4:612L21 antisense siNAuAuAcAuGGcAGGGuuccuTsT 1004 (594C) stab08 667 CCCCAAGAGAGUUAGCAAGUGCU941 33651 BIRC4:687L21 antisense siNA cAcuuGcuAAcucucuuGGTsT 1005 (669C)stab08 1345 CACUUGAGGUUCUGGUUGCAGAU 942 33652 BIRC4:1365L21 antisensesiNA cuGcAAccAGAAccucAAGTsT 1006 (1347C) stab08  94GCGAAAAGGUGGACAAGUCCUAU 935 33629 BIRC4:96U21 sense siNA stab09 BGAAAAGGUGGACAAGUCCUTT B 1007 314 GUGCUUUAGUUGUCAUGCAGCUG 936 33630BIRC4:316U21 sense siNA stab09 B GCUUUAGUUGUCAUGCAGCTT B 1008 353UGGAGACUCAGCAGUUGGAAGAC 937 33631 BIRC4:355U21 sense siNA stab09 BGAGACUCAGCAGUUGGAAGTT B 1009 358 ACUCAGCAGUUGGAAGACACAGG 938 33632BIRC4:360U21 sense siNA stab09 B UCAGCAGUUGGAAGACACATT B 1010 429AGUGCCACGCAGUCUACAAAUUC 939 33633 BIRC4:431U21 sense siNA stab09 BUGCCACGCAGUCUACAAAUTT B 1011 592 CGAGGAACCCUGCCAUGUAUAGU 940 33634BIRC4:594U21 sense siNA stab09 B AGGAACCCUGCCAUGUAUATT B 1012 667CCCCAAGAGAGUUAGCAAGUGCU 941 33635 BIRC4:669U21 sense siNA stab09 BCCAAGAGAGUUAGCAAGUGTT B 1013 1345 CACUUGAGGUUCUGGUUGCAGAU 942 33636BIRC4:1347U21 sense siNA stab09 B CUUGAGGUUCUGGUUGCAGTT B 1014  94GCGAAAAGGUGGACAAGUCCUAU 935 33637 BIRC4:114L21 antisense siNAAGGACUUGUCCACCUUUUCTsT 1015 (96C) stab10 314 GUGCUUUAGUUGUCAUGCAGCUG 93633638 BIRC4:334L21 antisense siNA GCUGCAUGACAACUAAAGCTsT 1016 (316C)stab10 353 UGGAGACUCAGCAGUUGGAAGAC 937 33639 BIRC4:373L21 antisense siNACUUCCAACUGCUGAGUCUCTsT 1017 (355C) stab10 358 ACUCAGCAGUUGGAAGACACAGG938 33640 BIRC4:378L21 antisense siNA UGUGUCUUCCAACUGCUGATsT 1018 (360C)stab10 429 AGUGCCACGCAGUCUACAAAUUC 939 33641 BIRC4:449L21 antisense siNAAUUUGUAGACUGCGUGGCATsT 1019 (431C) stab10 592 CGAGGAACCCUGCCAUGUAUAGU940 33642 BIRC4:612L21 antisense siNA UAUACAUGGCAGGGUUCCUTsT 1020 (594C)stab10 667 CCCCAAGAGAGUUAGCAAGUGCU 941 33643 BIRC4:687L21 antisense siNACACUUGCUAACUCUCUUGGTsT 1021 (669C) stab10 1345 CACUUGAGGUUCUGGUUGCAGAU942 33644 BIRC4:1365L21 antisense siNA CUGCAACCAGAACCUCAAGTsT 1022(1347C) stab10  94 GCGAAAAGGUGGACAAGUCCUAU 935 BIRC4:114121 antisensesiNA AGGAcuuGuccAccuuuucTT B 1023 (96C) stab19 314GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:334L21 antisense siNAGcuGcAuGAcAAcuAAAGcTT B 1024 (316C) stab19 353 UGGAGACUCAGCAGUUGGAAGAC937 BIRC4:373L21 antisense siNA cuuccAAcuGcuGAGucucTT B 1025 (355C)stab19 358 ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:378L21 antisense siNAuGuGucuuccAAcuGcuGATT B 1026 (360C) stab19 429 AGUGCCACGCAGUCUACAAAUUC939 BIRC4:449L21 antisense siNA AuuuGuAGAcuGcGuGGcATT B 1027 (431C)stab19 592 CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:612L21 antisense siNAuAuAcAuGGcAGGGuuccuTT B 1028 (594C) stab19 667 CCCCAAGAGAGUUAGCAAGUGCU941 BIRC4:687L21 antisense siNA cAcuuGcuAAcucucuuGGTT B 1029 (669C)stab19 1345 CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1365L21 antisense siNAcuGcAAccAGAAccucAAGTT B 1030 (1347C) stab19  94 GCGAAAAGGUGGACAAGUCCUAU935 BIRC4:114121 antisense siNA AGGACUUGUCCACCUUUUCTT B 1031 (96C)stab22 314 GUGCUUUAGUUGUCAUGCAGCUG 936 BIRC4:334L21 antisense siNAGCUGCAUGACAACUAAAGCTT B 1032 (316C) stab22 353 UGGAGACUCAGCAGUUGGAAGAC937 BIRC4:373L21 antisense siNA CUUCCAACUGCUGAGUCUCTT B 1033 (355C)stab22 358 ACUCAGCAGUUGGAAGACACAGG 938 BIRC4:378L21 antisense siNAUGUGUCUUCCAACUGCUGATT B 1034 (360C) stab22 429 AGUGCCACGCAGUCUACAAAUUC939 BIRC4:449L21 antisense siNA AUUUGUAGACUGCGUGGCATT B 1035 (431C)stab22 592 CGAGGAACCCUGCCAUGUAUAGU 940 BIRC4:612L21 antisense siNAUAUACAUGGCAGGGUUCCUTT B 1036 (594C) stab22 667 CCCCAAGAGAGUUAGCAAGUGCU941 BIRC4:687L21 antisense siNA CACUUGCUAACUCUCUUGGTT B 1037 (669C)stab22 1345 CACUUGAGGUUCUGGUUGCAGAU 942 BIRC4:1365L21 antisense siNACUGCAACCAGAACCUCAAGTT B 1038 (1347C) stab22 Uppercase = ribonucleotideu,c = 2-deoxy-2′-fluoro U, C T = thymidine B = inverted deoxy abasic s= phosphorothioate linkage A = deoxy Adenosine G = deoxy GuanosineG = 2′-O-methyl Guanosine A = 2′-O-methyl Adenosine

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at 3′- S/AS ends “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All linkages Usually AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′- — Usually S ends “Stab 5” 2′-fluoro Ribo — 1at 3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and 3′- — Usually Sends “Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′- — Usually S ends “Stab 8”2′-fluoro 2′-O- — 1 at 3′-end S/AS Methyl “Stab 9” Ribo Ribo 5′ and 3′-— Usually S ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11”2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′and 3′- Usually S ends “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually AS 1 at 3′-end “Stab 16” Ribo2′-O- 5′ and 3′- Usually S Methyl ends “Stab 17” 2′-O-Methyl 2′-O- 5′and 3′- Usually S Methyl ends “Stab 18” 2′-fluoro 2′-O- 5′ and 3′-Usually S Methyl ends “Stab 19” 2′-fluoro 2′-O- 3′-end S/AS Methyl “Stab20” 2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′- Usually S ends “Stab 24” 2′-fluoro* 2′-O- — 1 at3′-end S/AS Methyl* “Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end S/ASMethyl* “Stab 26” 2′-fluoro* 2′-O- — S/AS Methyl* “Stab 27” 2′-fluoro*2′-O- 3′-end S/AS Methyl* “Stab 28” 2′-fluoro* 2′-O- 3′-end S/AS Methyl*CAP = any terminal cap, see for example FIG. 10. All Stab 00-28chemistries can comprise 3′-terminal thymidine (TT) residues All Stab00-28 chemistries typically comprise about 21 nucleotides, but can varyas described herein. S = sense strand AS = antisense strand *Stab 23 hasa single ribonucleotide adjacent to 3′-CAP *Stab 24 and Stab 28 have asingle ribonucleotide at 5′-terminus *Stab 25, Stab 26, and Stab 27 havethree ribonucleotides at 5′-terminus p = phosphorothioate linkage

TABLE V Wait Time* Reagent Equivalents Amount Wait Time* DNA 2′-O-methylWait Time* RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl 186 233 μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec5 sec 5 sec Imidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents: Wait DNA/2′-O- Amount: DNA/ Wait Time* WaitTime* Time* Reagent methyl/Ribo 2′-O-methyl/Ribo DNA 2′-O-methyl RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole 70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl502/502/502 50/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

1. A chemically modified nucleic acid molecule, wherein: (a) the nucleicacid molecule comprises a sense strand and a separate antisense strand,each strand having one or more pyrimidine nucleotides and one or morepurine nucleotides; (b) each strand of the nucleic acid molecule isindependently 18 to 27 nucleotides in length; (c) an 18 to 27 nucleotidesequence of the antisense strand is complementary to a human XIAP RNAsequence comprising SEQ ID NO:1061; (d) an 18 to 27 nucleotide sequenceof the sense strand is complementary to the antisense strand andcomprises an 18 to 27 nucleotide sequence of the human RNA sequence; and(e) 50 percent or more of the nucleotides in at least one strandcomprise a 2-sugar modification, wherein the 2′-sugar modification ofany of the pyrimidine nucleotides differs from the 2′-sugar modificationof any of the purine nucleotides.
 2. The nucleic acid molecule of claim1, wherein 50 percent or more of the nucleotides in each strand comprisea 2′-sugar modification.
 3. The nucleic acid molecule of claim 1,wherein the 2′-sugar modification is selected from the group consistingof 2′-deoxy-2′-fluoro, 2′-O-methyl, and 2′-deoxy.
 4. The nucleic acid ofclaim 3, wherein the 2′-deoxy-2′-fluoro sugar modification is apyrimidine modification.
 5. The nucleic acid of claim 3, wherein the2′-deoxy sugar modification is a pyrimidine modification.
 6. The nucleicacid of claim 3, wherein the 2′-O-methyl sugar modification is apyrimidine modification.
 7. The nucleic acid molecule of claim 4,wherein said pyrimidine modification is in the sense strand, theantisense strand, or both the sense strand and antisense strand.
 8. Thenucleic acid molecule of claim 6, wherein said pyrimidine modificationis in the sense strand, the antisense strand, or both the sense strandand antisense strand.
 9. The nucleic acid molecule of claim 3, whereinthe 2′-deoxy sugar modification is a purine modification.
 10. Thenucleic acid molecule of claim 3, wherein the 2′-O-methyl sugarmodification is a purine modification.
 11. The nucleic acid molecule ofclaim 9, wherein the purine modification is in the sense strand.
 12. Thenucleic acid molecule of claim 10, wherein the purine modification is inthe antisense strand.
 13. The nucleic acid molecule of claim 1, whereinthe nucleic acid molecule comprises ribonucleotides.
 14. The nucleicacid molecule of claim 1, wherein the sense strand includes a terminalcap moiety at the 5′-end, the 3′-end, or both of the 5′- and 3′-ends.15. The nucleic acid molecule of claim 14, wherein the terminal capmoiety is an inverted deoxy abasic moiety.
 16. The nucleic acid moleculeof claim 1, wherein said nucleic acid molecule includes one or morephosphorothioate internucleotide linkages.
 17. The nucleic acid moleculeof claim 16, wherein one of the phosphorothioate internucleotidelinkages is at the 3′-end of the antisense strand.
 18. The nucleic acidmolecule of claim 1, wherein the 5′-end of the antisense strand includesa terminal phosphate group.
 19. The nucleic acid molecule of claim 1,wherein the sense strand, the antisense strand, or both the sense strandand the antisense strand include a 3′-overhang.
 20. A compositioncomprising the nucleic acid molecule of claim 1, in a pharmaceuticallyacceptable carrier or diluent.